Author: logancollins

The Virus Zoo: A Primer on Molecular Virology

July 27, 2022January 29, 2023By logancollins 4 Comments

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Human Immunodeficiency Virus (HIV)

Genome and Structure:

HIV’s genome is a 9.7 kb linear positive-sense ssRNA.¹ There is a m⁷G-cap (specifically the standard eukaryotic m⁷GpppG as added by the host’s enzymes) at the 5’ end of the genome and a poly-A tail at the 3’ end of the genome.² The genome also has a 5’-LTR and 3’-LTR (long terminal repeats) that aid its integration into the host genome after reverse transcription, that facilitate HIV genetic regulation, and that play a variety of other important functional roles. In particular, it should be noted that the integrated 5’UTR contains the HIV promoter called U3.^3,4

HIV’s genome translates three polyproteins (as well as several accessory proteins). The Gag polyprotein contains the HIV structural proteins. The Gag-Pol polyprotein contains (within its Pol component) the enzymes viral protease, reverse transcriptase, and integrase. The Gag-Pol polyprotein is produced via a –1 ribosomal frameshift at the end of Gag translation. Because of the lower efficiency of this frameshift, Gag-Pol is synthesized 20-fold less frequently than Gag.⁵ The frameshift’s mechanism depends upon a slippery heptanucleotide sequence UUUUUUA and a downstream RNA secondary structure called the frameshift stimulatory signal (FSS).⁶ This FSS controls the efficiency of the frameshift process.

The HIV RNA genome undergoes alternative splicing to produce the rest of the viral proteins. One splicing event produces an RNA that separately encodes the Vpu protein and the Env protein (also called gp160).^6–8 A mechanism called ribosome shunting is used to transition from Vpu’s open reading frame to Env’s open reading frame. The Env protein contains the gp41 and gp120 proteins. Env is post-translationally cleaved into gp41 and gp120 by a host furin enzyme in the endoplasmic reticulum.⁹ It is important to note that Env is also heavily glycosylated post-translationally to help HIV evade the immune system. Several other complex splicing events lead to the production of RNAs encoding Tat, Rev, Nef, Vif, and Vpr.

HIV viral protease cleaves the Gag polyprotein and thus produces structural proteins including the capsid protein CA (also called p24), the matrix protein MA (also called p17), the nucleocapsid protein NC (also called p7), and the p6 peptide.¹⁰ The HIV core capsid is shaped like a truncated cone and consists of about 1500 CA monomers. Most of the CA proteins assemble into hexamers, but a few pentamers are present. The pentamers help give the core capsid its conical morphology by providing extra curvature near the top and bottom. Each core capsid contains two copies of the HIV genomic RNA, complexed with NC protein. Reverse transcriptase, integrase, and viral accessory proteins are also held within the core capsid. HIV’s core capsid is packaged into a lipid envelope that bears gp41-gp120 glycoprotein heterodimers. The MA protein forms a layer between the core capsid and the envelope.

Accessory proteins Vpu, Tat, Rev, Nef, Vif, and Vpr facilitate a variety of functions. Vpu induces degradation of CD4 proteins within the endoplasmic reticulum of host CD4+ T cells. It does this by using its cytosolic domain as a molecular adaptor between CD4 and a ubiquitin ligase (which subsequently triggers proteosomal degradation of the CD4).¹¹ The reason that Vpu does this is to prevent HIV superinfection wherein two different types of HIV might infect the same cell and interfere with each other. This is an example of competition between viruses.¹² Vpu also enhances release of HIV virions from infected cells by using its cytosolic domain to inhibit a host protein called tetherin (also known as BST-2).¹¹ Without Vpu, tetherin would bind the viral envelope to the cell surface as well to other HIV virus particles, impeding release.

Tat, also called the viral transactivator protein, is necessary for efficient transcriptional elongation of the HIV genome after integration into the host DNA.¹³ Tat binds the viral transactivation response element (TAR), a structured RNA motif present at the beginning of the HIV transcripts. It then recruits protein positive transcription elongation factor b (P-TEFb). This allows P-TEFb to phosphorylate certain residues in the C-terminal domain of RNA polymerase II, stimulating transcriptional elongation. Tat also recruits several of the host cell’s histone acetyltransferases to the viral 5’-LTR so as to open the chromatin around the U3 promoter and related parts of the integrated HIV genome.^3,4 Finally, Tat is secreted from infected cells¹⁴ and acts as an autocrine and paracrine signaling molecule.⁴ It inhibits antigen-specific lymphocyte proliferation, stimulates expression of certain cytokines and cytokine receptors, modulates the activities of various host cell types, causes neurotoxicity in the brain, and more.

Rev facilitates nuclear export of the unspliced and singly spliced HIV RNAs by binding to a sequence located in the Env coding region called the Rev response element (RRE).¹³ The Rev protein forms a dimer upon binding to the RRE and acts as an adaptor, binding a host nuclear export factor called CRM1. Rev is also known to form higher-order oligomers via cooperative multimerization of the RNA-bound dimers.

Nef is a myristoylated protein that downregulates certain host T cell proteins and thereby increases production of virus. Nef is localized to the cytosol and the plasma membrane. It specifically inhibits CD4, Lck, CTLA-4, and Bad.¹⁵ Downregulating CD4 contributes to the prevention of superinfection that also occurs with Vpu’s inhibition of CD4. Nef induces endocytosis of plasma membrane Lck protein and traffics it to recycling endosomes and the trans-Golgi network. At these intracellular compartments, Lck signals for Ras and Erk activation, which triggers IL-2 production. IL-2 causes T cells to grow and proliferate, leading to more T cells that HIV can infect and leading to activation of the machinery HIV needs to replicate itself within infected T cells. Nef triggers lysosomal degradation of CTLA-4. This is because CTLA-4 can serve as an off-switch for T cells, which would lead to inhibition of HIV replication if active. Nef inactivates the Bad protein via phosphorylation. Bad participates in apoptotic cascades, so Nef prevents apoptosis of the infected host cell in this way.

Vif forms a complex with the host antiviral proteins APOBEC3F and APOBEC3G and induces their ubiquitination and subsequent degradation by the proteosome.¹⁶ It also may inhibit these proteins through other mechanisms. APOBEC3F and APOBEC3G are cytidine deaminases that hypermutate the negative-sense strand of HIV cDNA, leading to weak or nonviable viruses.¹⁷ These proteins also interfere with reverse transcription by blocking tRNALys3 from binding to the HIV RNA 5’UTR (tRNALys3 usually acts as a primer to initiate reverse transcription of the HIV genome).¹⁸

Vpr facilitates nuclear import of the HIV pre-integration complex.¹⁹ The pre-integration complex consists of viral cDNA and associated proteins (uncoating and reverse transcription have already occurred at this stage). Vpr binds the pre-integration complex and recruits host importins to enable nuclear import. It may further enhance nuclear import through interactions with some of the nuclear pore proteins. In addition to nuclear import, Vpr has several more functions: it acts as a coactivator (along with other proteins) of the HIV 5’UTR’s U3 promoter, might influence NF-κB regulation, may modulate apoptotic pathways, and arrests the cell cycle at the G₂ stage.

Life cycle:

CD4+ T cells represent the primary targets of HIV, though the virus is also capable of infecting other cell types such as dendritic cells.²⁰ HIV infects CD4+ T cells through binding its gp120 glycoprotein to the CD4 receptor and the CCR5 coreceptor or the CXCR4 coreceptor.¹⁰ This triggers fusion of the viral envelope with the plasma membrane and allows the core capsid to enter the cytosol.

HIV’s core capsid is transported by motor proteins along microtubules to dock at nuclear pores. The nuclear pore complex has flexible cytosolic filaments composed primarily of the Nup358 protein, which interacts with the core capsid.²¹ These interactions guide the narrow end of the core capsid into the nuclear pore’s central channel. Next, the core capsid interacts with the central channel’s unstructured phenylalanine-glycine (FG) repeats that exist in a hydrogel-like liquid phase. As the core capsid translocates through the central pore, it binds the Nup153 protein, a component of the nuclear pore complex’s basket. Finally, many copies of the nucleoplasmic CPSF6 protein coat the core capsid and escort it towards its genomic site of integration. It is thought that the reverse transcription process begins inside of the core capsid at this point, leading to cDNA synthesis.^21,22 Buildup of newly made cDNA within the core capsid likely results in pressure that helps rupture the capsid structure, facilitating uncoating.

Tetrameric HIV integrase binds both of the viral LTRs and facilitates integration of the cDNA into the host genome.²³ Though integration sites vary widely, they are not entirely random. Host chromatin structure and other factors influence where the viral cDNA integrates.²⁴ Transcription of HIV RNAs can then proceed from the U3 promoter with the aid of the Tat protein and host factors. As described earlier, a series of RNA splicing events produce the various RNAs necessary to synthesize all of the different HIV proteins and polyproteins.

Env protein is trafficked to the cell membrane through the secretory pathway. It is cleaved by a host furin enzyme into gp41 and gp160 components during its time in the endoplasmic reticulum.⁹ Gag and Gag-Pol polyproteins are expressed cytosolically. Since Gag is post-translationally modified by amino-terminal myristoylation, it anchors to the cell membrane by inserting its myristate tail into the lipid bilayer.²⁵ Gag and a smaller number of Gag-Pol accumulate on the inner membrane surface and incorporate gp41-gp160 complexes. NC domains in the Gag proteins bind and help package the two copies of HIV genomic RNA. The p6 region of the Gag protein (located at the C-terminal end) then recruits host ESCRT-I and ALIX proteins, which subsequently sequester host ESCRT-III and VPS4 complexes to drive budding and membrane scission, releasing virus into the extracellular space. After this, the HIV viral protease (from within the Gag-Pol polyprotein) cleaves the Gag and Gag-Pol polyproteins into their constituent proteins, facilitating maturation of the released HIV particles.

SARS-CoV-2

Genome and Structure:

The SARS-CoV-2 genome consists of about 30 kb of linear positive-sense ssRNA. There is a m⁷G-cap (specifically m⁷GpppA₁) at the 5’ end of the genome and a 30-60 nucleotide poly-A tail at the 3’ end of the genome. These protective structures minimize exonuclease degradation.²⁶ The genome also has a 5’ UTR and a 3’ UTR which contain sequences that aid in transcriptional regulation and in packaging. The SARS-CoV-2 genome directly translates two partially overlapping polyproteins, ORF1a and ORF1b. There is a –1 ribosomal frameshift in ORF1b relative to ORF1a. Within the polyproteins, two self-activating proteases (Papain-like protease PLpro and 3-chymotrypsin-like protease 3CLpro) perform cleavage events that lead to the generation of the virus’s 16 non-structural proteins (nsps). It should be noted that the 3CLpro is also known as the main protease or M^pro. The coronavirus also produces 4 structural proteins, but these are not translated until after the synthesis of corresponding subgenomic RNAs via the viral replication complex. To create these subgenomic RNAs, negative-sense RNA must first be made and then undergo conversion back to positive-sense RNA for translation. Genes encoding the structural proteins are located downstream of the ORF1b section.

SARS-CoV-2’s four structural proteins include the N, E, M, and S proteins. Many copies of the N (nucleocapsid) protein bind the RNA genome and organize it into a helical ribonucelocapsid complex. The complex undergoes packaging into the viral envelope during coronavirus budding. Interactions between the N protein and the other structural proteins may facilitate this packaging process. The N protein also inhibits host immune responses by antagonizing viral suppressor RNAi and by blocking the signaling of interferon production pathways.²⁷

The transmembrane E (envelope) protein forms pentamers and plays a key but poorly understood role in the budding of viral envelopes into the endoplasmic reticulum Golgi intermediate compartment (ERGIC).^28–30 Despite its importance in budding, mature viral particles do not incorporate very many E proteins into their envelopes. One of the posttranslational modifications of the E protein is palmitoylation. This aids subcellular trafficking and interactions with membranes. E protein pentamers also act as ion channels that alter membrane potential.^31,32 This may lead to inflammasome activation, a contributing factor to cytokine storm induction.

The M (membrane) protein is the most abundant protein in the virion and drives global curvature in the ERGIC membrane to facilitate budding.^30,33 It forms transmembrane dimers that likely oligomerize to induce this curvature.³⁴ The M protein also has a cytosolic (and later intravirion) globular domain that likely interacts with the other structural proteins. M protein dimers also induce local curvature through preferential interactions with phosphatidylserine and phosphatidylinositol lipids.^29,30 M proteins help sequester S proteins into the envelopes of budding viruses.³⁵

The S (spike) protein of SARS-CoV-2 has been heavily studied due to its central roles in the infectivity and immunogenicity of the coronavirus. It forms a homotrimer that protrudes from the viral envelope and is heavily glycosylated. It binds the host’s ACE2 receptor (angiotensin-converting enzyme 2 receptor) and undergoes conformational changes to promote viral fusion.³⁶ The S protein undergoes cleavage into S1 and S2 subunits by the host’s furin protease during viral maturation.^37,38 This enhances SARS-CoV-2 entry into lung cells and may partially explain the virus’s high degree of transmissibility. The S1 fragment contains the receptor binding domain (RBD) and associated machinery while the S2 fragment facilitates fusion. Prior to cellular infection, most S proteins exist in a closed prefusion conformation where the RBDs of each monomer are hidden most of the time.³⁹ After the S protein binds ACE2 during transient exposure of one of its RBDs, the other two RBDs quickly bind as well. This binding triggers a conformational change in the S protein that loosens the structure, unleashing the S2 fusion component and exposing another proteolytic cleavage site called S2’. Host transmembrane proteases such as TMPRSS2 cut at S2’, causing the full activation of the S2 fusion subunit and the dramatic elongation of the S protein into the postfusion conformation. This results in the viral envelope fusing with the host membrane and uptake of the coronavirus’s RNA into the cell.

The 16 nsps of SARS-CoV-2 play a variety of roles. For instance, nsp1 shuts down host cell translation by plugging the mRNA entry channel of the ribosome, inhibiting the host cell’s immune responses and maximizing viral production.^40,41 Viral proteins still undergo translation because a conserved sequence in the coronavirus RNA helps circumvent the blockage through a poorly understood mechanism. The nsp5 protein is the protease 3CLpro.⁴² The nsp3 protein contains several subcomponents, including the protease PLpro. The nsp12, nsp7, and nsp8 proteins come together to form the RNA-dependent RNA polymerase (RdRp) that replicates the viral genome.^42,43 The nsp2 protein is likely a topoisomerase which functions in RNA replication. The nsp4 and nsp6 proteins as well as certain subcomponents of nsp3 restructure intracellular host membranes into double-membrane vesicles (DMVs) which compartmentalize viral replication.⁴⁴

Beyond the 4 structural proteins and 16 nsps of SARS-CoV-2, the coronaviral genome also encodes some poorly understood accessory proteins including ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF9b.⁴⁵ These accessory proteins are non-essential for replication in vitro, but they are thought to be required for the virus’s full degree of virulence in vivo.

Life cycle:

As mentioned, SARS-CoV-2 infects cells by first binding a S protein RBD to the ACE2 receptor. This triggers a conformational change that elongates the S protein’s structure and reveals the S2 fusion fragment, facilitating fusion of the virion envelope with the host cell membrane.³⁹ Cleavage of the S’ site by proteases like TMPRSS2 aid this change from the prefusion to postfusion configurations. Alternatively, SARS-CoV-2 can enter the cell by binding to ACE2, undergoing endocytosis, and fusing with the endosome to release its genome (as induced by endosomal cathepsin proteases).⁴⁵ After release of the SARS-CoV-2 genome into the cytosol, the N protein disassociates and allows translation of ORF1a and ORF1b, producing polyproteins which are cleaved into mature proteins by the PLpro and 3CLpro proteases as discussed earlier.

The RdRp complex synthesizes negative-sense full genomic RNAs as well as negative-sense subgenomic RNAs. In the latter case, discontinuous transcription is employed, a process by which the RdRp jumps over certain sections of the RNA and initiates transcription separately from the rest of the genome.⁴⁶ The negative-sense RNAs are subsequently converted back into positive-sense full genomic RNAs and positive-sense subgenomic RNAs. The subgenomic RNAs are translated to make structural proteins and some accessory proteins.⁴⁵

As described earlier, the nsp4, nsp6, and parts of nsp3 proteins remodel host endoplasmic reticulum (ER) to create DMVs.⁴⁵ These DMVs are the site of the coronaviral genomic replication and serve to shield the viral RNA and RdRp complex from cellular innate immune factors. DMVs cluster together and are continuous with the ER mostly through small tubular connections. After replication, the newly synthesized coronavirus RNAs undergo export into the cytosol through molecular pore complexes that span both membranes of the DMVs.⁴⁷ These molecular pore complexes are composed of nsp3 domains and possibly other viral and/or host proteins.

Newly replicated SARS-CoV-2 genomic RNAs complex with N proteins to form helical nucleocapsids. To enable packaging, the nucleocapsids interact with M protein cytosolic domains which protrude at the ERGIC.⁴⁸ M proteins, E proteins, and S proteins are all localized to the ERGIC membrane. The highly abundant M proteins induce curvature of the membrane to facilitate budding. As mentioned, E proteins also play essential roles in budding, but the mechanisms are poorly understood. Once the virions have budded into the ERGIC, they are shuttled through the Golgi via a series of vesicles and eventually secreted out of the cell.

Adeno-associated virus (AAV)

Genome and Structure:

AAV genomes are about 4.7 kb in length and are composed of ssDNA. Inverted terminal repeats (ITRs) form hairpin structures at ends of the genome. These ITR structures are important for AAV genomic packaging and replication. Rep genes (encoded via overlapping reading frames) include Rep78, Rep68, Rep52, Rep40.⁴⁹ These proteins facilitate replication of the viral genome. As a Dependoparvovirus, additional helper functions from adenovirus (or certain other viruses) are needed for AAVs to replicate.

AAV capsids are about 25 nm in diameter. Cap genes include VP1, VP2, VP3 and are transcribed from overlapping reading frames.⁵⁰ The VP3 protein is the smallest capsid protein. The VP2 protein is the same as VP3 except that it includes an N-terminal extension with a nuclear localization sequence. The VP1 protein is the same as VP2 except that it includes a further N-terminal extension encoding a phospholipase A2 (PLA2) that facilitates endosomal escape during infection. In the AAV capsid, VP1, VP2, and VP3 are present at a ratio of roughly 1:1:10. It should be noted that this ratio is actually the average of a distribution, not a fixed number.

Frame-shifted start codons in the Cap gene region transcribe AAP (assembly activating protein) and MAAP (membrane associated accessory protein). These proteins help facilitate packaging and other aspects of the AAV life cycle.

Life cycle:

There are a variety of different AAV serotypes (AAV2, AAV6, AAV9, etc.) that selectively infect certain tissue types. AAVs bind to host cell receptors and are internalized by endocytosis. The particular receptors involved can vary depending on the AAV serotype, though some receptors are consistent across many serotypes. Internalization occurs most often via clathrin-coated pits, but some AAVs are internalized by other routes such as macropinocytosis or the CLIC/GEEC tubulovesicular pathway.⁵¹

After endocytosis, conformational changes in the AAV capsid lead to exposure of the PLA2 VP1 domain, which facilitates endosomal escape. The AAV is then transported to the nucleus mainly by motor proteins on cytoskeletal highways. It enters via nuclear pores and finishes uncoating its genome.

AAV genomes initiate replication using the ends of their ITR hairpins as primers. This leads to a series of complex steps involving strand displacement and nicking.⁴⁹ In the end, new copies of the AAV genome are synthesized. The Rep proteins are key players in this process. It is important to realize that AAVs can only replicate in cells which have also been infected by adenovirus or similar helper viruses (this is why they are called “adeno-associated viruses”). Adenoviruses provide helper genes encoding proteins (e.g. E4, E2a, VA) that are vital for the successful completion of the AAV life cycle. After new AAV capsids have assembled from VP1, VP2, and VP3 and once AAV genomes have been replicated, the ssDNA genomes are threaded into the capsids via pores at their five-fold vertices.

AAVs are nonpathogenic, though a large fraction of people possess antibodies against at least some serotypes, so exposure to them is fairly common.

Adenovirus

Genome and Structure:

Adenovirus genomes are about 36 kb in size and are composed of linear dsDNA. They possess inverted terminal repeats (ITRs) which help facilitate replication and other functions. These genomes contain a variety of transcriptional units which are expressed at different times during the virus’s life cycle.⁵² E1A, E1B, E2A, E2B, E3, and E4 transcriptional units are expressed early during cellular infection. Their proteins are involved in DNA replication, transcriptional regulation, and suppression of host immune responses. The L1, L2, L3, L4, and L5 transcriptional units are expressed later in the life cycle. Their products include most of the capsid proteins as well as other proteins involved in packaging and assembly. Each transcriptional unit can produce multiple mRNAs through the host’s alternative splicing machinery.

The capsid of the adenovirus is about 90 nm in diameter and consists of three major proteins (hexon, penton, and fiber proteins) as well as a variety of minor proteins and core proteins. Hexon trimer is the most abundant protein in the capsid, the pentameric pentons occur at the vertices, and trimeric fibers are positioned on top of the pentons.⁵³ The fibers point outwards from the capsid and end in knob domains which bind to cellular receptors. In Ad5, a commonly studied type of adenovirus, the fiber knob primarily binds to the coxsackievirus and adenovirus receptor (CAR). That said, it should be noted that Ad5’s fiber knob can also bind to alternative receptors such as vascular cell adhesion molecule 1 and heparan sulfate proteoglycans.

Minor capsid proteins include pIX, pIIIa, pVI, and pVIII. The pIX protein interlaces between hexons and helps stabilize the capsid. Though pIX is positioned in the crevices between the hexons, it is still exposed to the outside environment. By contrast, the pIIIa, pVI, and pVIII proteins bind to the inside of the capsid and contribute further structural stabilization. When the adenovirus is inside of the acidic endosome during infection, conformational changes in the capsid release the pVI protein, which facilitates endosomal escape through membrane lytic activity.

Adenovirus core proteins include pV, pVII, protein μ (also known as pX), adenovirus proteinase (AVP), pIVa2, and terminal protein (TP).⁵⁴ The pVII protein has many positively-charged arginine residues and so functions to condense the viral DNA. The pV protein bridges the core with the capsid through interactions with pVII and with pVI. AVP cleaves various adenoviral proteins (pIIIa, TP, pVI, pVII, pVIII, pX) to convert them to their mature forms.⁵⁵ The pIVa2 and pX proteins interact with the viral DNA and may play roles in packaging or replication. TP binds to the ends of the genome and is essential for localizing the viral DNA in the nucleus and for viral replication.

Life Cycle:

Adenovirus infects cells by binding its fiber knob to cellular receptors such as CAR (in the case of Ad5). The penton then binds certain α_v integrins, positioning the viral capsid for endocytosis.⁵⁶ When the endosome acidifies, the adenovirus capsid partially disassembles, fibers and pentons fall away, and pVI is released.⁵⁷ The pVI protein’s membrane lytic activity facilitates endosomal escape. Partially disassembled capsids then undergo dynein-mediated transport along microtubules and dock at the entrance to nuclear pores. The capsids further disassemble and releases DNA through the nuclear pore. This DNA remains complexed with pVII after it enters the nucleus.

Adenoviral transcription is initiated by the E1A protein, inducing expression of early genes.⁵⁸ This subsequently leads to expression of the E2, E3, and E4 transcriptional units, which help the virus escape immune responses. This cascade leads to expression of the L1, L2, L3, L4, and L5 transcriptional units, which mainly synthesize viral structural proteins and facilitate capsid assembly.

In the nucleus, adenovirus genomes replicate within dense complexes of protein that can be seen as spots via fluorescence microscopy. Replication begins at the ITRs and is primed by TP.⁵⁹ Several more viral proteins and host proteins also aid the initiation of replication. Nontemplate strands are displaced during replication but may reanneal and act as template strands later. Adenovirus DNA binding protein and adenovirus DNA polymerase play important roles in replication. Once the genome has been replicated, TP undergoes cleavage into its mature form, signaling for packaging of new genomes.

The adenoviral capsid assembly and maturation process occurs in the nucleus.⁵⁸ Once enough assembled adenoviruses have accumulated, they rupture the nuclear membrane using adenoviral death protein and subsequently lyse the cell, releasing adenoviral particles.

Herpes Simplex Virus 1 (HSV-1)

Genome and Structure:

HSV-1 genomes are about 150 kb in size and are composed of linear dsDNA. These genomes include a unique long (U_L) region and a unique short (U_S) region.⁶⁰ The U_L and U_S regions are both flanked by their own inverted repeats. The terminal inverted repeats are called TR_L and TR_S while the internal inverted repeats are called IR_L and IR_S. HSV-1 contains approximately 80 genes, though the complexity of its genomic organization makes an exact number of genes difficult to obtain. As with many other viruses, HSV-1 genomes encode early, middle, and late genes. The early genes activate and regulate transcription of the middle and late genes. Middle genes facilitate genome replication and late genes mostly encode structural proteins.

The diameter of HSV-1 ranges around 155 nm to 240 nm.⁶¹ Its virions include an inner icosahedral capsid (with a 125 nm diameter) surrounded by tegument proteins which are in turn enveloped by a lipid membrane containing glycoproteins.

HSV-1’s icosahedral capsid consists of a variety of proteins. Some of the most important capsid proteins are encoded by the UL19, UL18, UL38, UL6, UL17, and UL25 genes.⁶² The UL19 gene encodes the major capsid protein VP5, which forms pentamers and hexamers for the capsid. These VP5 pentamers and hexamers are glued together by triplexes consisting of two copies of VP23 (encoded by UL18) and one copy of VP19C (encoded by UL38).⁶³ The UL6 gene encodes the protein that makes up the portal complex, a structure used by HSV-1 to release its DNA during infection. Each HSV-1 capsid has a single portal (composed of 12 copies of the portal protein) located at one of the vertices. UL17 and UL25 encode additional structural proteins that stabilize the capsid by binding on top of the other vertices. These two proteins also serve as a bridge between the capsid core and the tegument proteins.

The tegument of HSV-1 contains dozens of distinct proteins. Some examples include pUL36, pUL37, pUL7, and pUL51 proteins. The major tegument proteins are pUL36 and pUL37. The pUL36 protein binds on top of the UL17-UL25 complexes at the capsid’s vertices.⁶⁴ The pUL37 protein subsequently associates with pUL36. The pUL51 protein associates with cytoplasmic membranes in infected cells and recruits the pUL7 protein.⁶⁵ This pUL51-pUL7 interaction is important for HSV-1 assembly. HSV-1 has many more tegument proteins which play various functional roles.

HSV-1’s envelope contains up to 16 unique glycoproteins. Four of these glycoproteins (gB, gD, gH, and gL) are essential for viral entry into cells.⁶⁶ The gD glycoprotein first binds to one of its cellular receptors (nectin-1, herpesvirus entry mediator or HVEM, or 3-O-sulfated heparan sulfate). This binding event triggers a conformational change in gD that allows it to activate the gH/gL heterodimer. Next, gH/gL activate gB which induces fusion of HSV-1’s envelope with the cell membrane. Though the remaining 12 envelope glycoproteins are poorly understood, it is thought that they also play roles that influence cellular tropism and entry.

Life cycle:

After binding to cellular receptors via its glycoproteins, HSV-1 induces fusion of its envelope with the host cell membrane.⁶⁷ The capsid is trafficked to nuclear pores via microtubules. Since the capsid is too large to pass through a nuclear pore directly, the virus instead ejects its DNA through the pore via the portal complex.⁶⁸

HSV-1 replicates its genome and assembles its capsids in the nucleus. But the assembled capsids are again too large to exist the nucleus through nuclear pores. To overcome this issue, HSV-1 first buds via the inner nuclear membrane into the perinuclear cleft (the space between nuclear membranes), acquiring a primary envelope.⁶⁷ This process is driven by a pair of proteins (pUL34 and pUL31) which together form the nuclear egress complex. Next, the primary envelope fuses with the outer nuclear membrane, releasing the assembled capsids into the cytosol.

To acquire its final envelope, the HSV-1 capsid likely buds into the trans-Golgi network or into certain tubular vesicular organelles.⁶⁹ These membrane sources contain the envelope proteins of the virus as produced by transcription and various secretory pathways. One player is the pUL51 tegument protein that starts associated with the membrane into which the virus buds. The interaction between pUL51 and pUL7 helps facilitate recruitment of the capsid to the membrane. (Capsid envelopment is also coupled in many other ways to formation of the outer tegument). The enveloped virion eventually undergoes trafficking through the secretory system and eventually is packaged into exosomes that fuse with the cell membrane and release completed virions into the extracellular environment.

In humans, HSV-1 infects the epithelial cells first and produces viral particles.⁷⁰ It subsequently enters the termini of sensory neurons, undergoes retrograde transport into the brain, and remains in the central nervous system in a dormant state. During periods of stress in the host, the virus is reactivated and undergoes anterograde transport to infect epithelial cells once again.

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Guide to the Structure and Function of the Adenovirus Capsid

April 27, 2022April 27, 2022By logancollins 1 Comment

PDF version: Guide to the Structure and Function of the Adenovirus Capsid

For this guide, I will explain the fundamental biology of adenovirus capsid proteins with an emphasis on the context of gene therapy. While the guide is meant primarily for readers with an interest in applying adenovirus to gene therapy, it will not include much discussion of the techniques and technologies involved in engineering adenoviruses for such purposes. If you are interested in learning more about adenovirus engineering, you may enjoy my review paper “Synthetic Biology Approaches for Engineering Next-Generation Adenoviral Gene Therapies” [1]. Here, I will focus mostly on the capsid of human adenovirus serotype 5 (Ad5) since it is the most commonly used type of adenovirus employed in gene therapy research, but I will occasionally describe other types of adenoviruses when necessary. Many of the presented concepts remain the same or similar across other types of adenoviruses.

The adenovirus consists of an icosahedral protein capsid enclosing a double-stranded DNA (dsDNA) genome. It possesses 12 fiber proteins which protrude from the capsid and helps to facilitate cellular transduction. Adenoviruses are nonenveloped and approximately 90 nm in diameter (not including the fibers). The Ad5 genome is about 36 kb in size. Major capsid proteins of the adenovirus include the hexon, penton, and fiber. The minor capsid proteins are protein IIIa, protein VI, protein VIII, and protein IX. Inside the capsid, there are core proteins including protein V, protein VII, protein μ (also known as protein X), adenovirus proteinase (AVP), protein IVa2, and terminal protein (TP) [2]. There are also many proteins expressed during adenovirus infection which are not incorporated into mature capsids, including the E1A proteins (289R, 243R, 217R, 171R, and 55R), the E1B proteins (52k and 55k), the adenoviral DNA polymerase, and more [3].

Ad5’s genome contains a variety of transcriptional units which are expressed at different times during the viral life cycle [3]. The E1A, E1B, E2A, E2B, E3, and E4 transcriptional units are expressed early during cellular infection. Their proteins are involved in DNA replication, transcriptional regulation, and suppression of host immune responses. The L1, L2, L3, L4, and L5 transcriptional units are expressed later in the life cycle. Their products include most of the capsid proteins as well as other proteins involved in packaging and assembly. Each transcriptional unit can produce multiple mRNAs through the host’s alternative splicing machinery.

Major capsid proteins

Hexon

Adenovirus hexon represents the main structural component of the capsid. It is encoded as one of the products of the Ad5 L3 gene. Each capsid contains 240 trimers of the hexon protein (720 monomers) and each facet of the icosahedron consists of 12 trimers [4]. The lower part of each hexon monomer consists of two eight-stranded β barrels linked by a β-sheet. The eight-stranded β-barrels are known as jellyroll domains. In between the β-strands, long loops are present. These loops contain the seven hypervariable regions (HVRs) of the hexon, which differ in sequence composition between distinct adenovirus types. The loops form the upper portion of each hexon. HVR1 of Ad5 includes a 32-residue acidic loop which might be involved in neutralizing host defensins. The valley between the loop towers of Ad5 has been shown to interact with coagulation factors as well as to bind to the CD46 cellular receptor as an alternative cell entry mechanism.

Here, the structure of the Ad5 hexon trimer is shown from a side view and from a top view (PDB 1P30). All β-sheets are red, α-helices are cyan, and loops are magenta. Jellyroll domains are visible at the base of the side view and the HVR loops can be seen in the upper half of the side view. In the top view, the hexagonal shape of the hexon is clearly visible. The N- and C- termini are both located near the bottom of the hexon (adjacent to the inside of the virion). Some disordered regions are shown as dashed lines.

Penton

The 12 pentons serve to fill pentagonal gaps within the icosahedral capsid (which arise due to the geometry of the hexons) [4]. Penton is encoded as one of the products of the Ad5 L2 gene. Each penton also acts as a base onto which a fiber protein is anchored. Adenovirus pentons are pentamers, with each monomeric subunit consisting of a single jellyroll domain for the lower part and both a hypervariable loop and a variable loop at the top. In Ad5 and many other human adenoviruses, the penton hypervariable loop includes an RGD amino acid sequence. RGD is both an α_v integrin binding motif and is a target for adenovirus neutralization by the enteric defensin HD5. Importantly, the penton’s RGD motif is essential for cellular transduction into clathrin-coated pits [5]. RGD may also play some role in endosomal escape. The other penton variable loop (distinct from the hypervariable loop) is poorly understood from a functional standpoint. Both the hypervariable loop and the variable loop might serve as decent sites for sequence modification in the context of gene therapy vectors. The penton N-terminal domain consists of approximately 50 amino acid sequence which extends into the inside of the adenovirus virion. This sequence is mostly disordered except for the part nearest to the jellyroll domain (residues 37-51 in Ad5), which interacts with two copies of protein IIIa.

Here, the structure of the Ad5 penton is shown from side and top views (PDB 3IZO). Coloration is by subunit. In the side view, the intravirion N-terminal domains are visible at the bottom, the jellyroll domains can be seen as the groups of β-sheets in the middle, and the loops are present at the upper region. The top view clearly illustrates the pentagonal symmetry of the penton. It should be noted that, in this structure, some of the loops are missing due to the difficulty of reconstructing them at high resolution. Of special relevance here is that the loop with the RGD sequence should be located at the top of the penton (in the gap between the uppermost α-helix and a nearby loop which both terminate prematurely).

Fiber

Ad5’s 12 trimeric fibers are anchored onto the tops of the pentons [4]. They are encoded as a product of the L5 gene. These fibers initiate cellular transduction through binding of the knob domain to cellular receptors. The primary receptor for Ad5 is the coxsackievirus and adenovirus receptor (CAR). That said, it should be noted that Ad5’s fiber knob can also bind to alternative receptors such as vascular cell adhesion molecule 1 and heparan sulfate proteoglycans. For Ad5, the fiber is about 37 nm in length, but other adenoviruses can have shorter or longer fibers [6]. Fibers consist of an N-terminal tail domain, a shaft domain, and a C-terminal knob (also called head) domain [4]. The three N-terminal tails anchor into some of the clefts between penton monomers, likely via a hydrophobic ring region. The shaft consists of a structure known as a trimeric β-spiral. Shaft flexibility plays a role in cellular transduction by facilitating interaction of the penton with its integrin receptor after binding of the knob to CAR. Many adenovirus fibers are known to have hinges at the third β-repeat from the N-terminal tail domain [7]. These hinges arise from an insertion of a few extra amino acids within the third β-repeat which disrupts its structure and allows for it to flex. The C-terminal knob domain consists of an antiparallel β-sandwich and is responsible for trimerization of the fiber [4]. Its C-termini are oriented back towards the capsid of the adenovirus.

Here, part of the structure of an Ad2 fiber is shown from two perspective views (PDB 1QIU). Though there are structures of the Ad5 fiber components available, only the above Ad2 fiber structure has been assembled into a complex with and made publicly available. The Ad2 fiber is highly similar to the Ad5 fiber. Both Ad5 and Ad2 fibers have 22 β-repeats. Only a few β-repeats are included in the above structures, but that should be enough to grant an intuitive understanding of the general fiber organization.

Minor capsid proteins

Protein IX

Ad5 protein IX (pIX) is a 140 amino acid protein found nestled between hexons which confers greater thermostability to the capsid relative to mutants lacking pIX [4]. There are 240 copies of pIX in the capsid. It has an N-terminal domain, a rope domain, and a C-terminal domain. The N-terminal domains of three pIX monomers interlace to form a triskelion structure in the valleys between some of the hexons. The rope domain (also called linker domain) is often disordered and connects the N- and C-terminal domains. The C-terminal domain is an α-helix which forms a coiled-coil structure with the helices of other copies of pIX monomer. This coiled coil consists of four α-helices (three parallel and one antiparallel), each from a different pIX monomer. Four triskelions and three α-helix bundles are present in each icosahedral facet of the capsid. It should be noted that some of the triskelions take on slightly different structural features depending on which hexons they are associated with within a given facet [8]. Though all of the C-termini of pIX are exposed on the capsid surface, they can still be described as resting within crevices between hexons. Because of this, spacer peptides are usually necessary when engineering Ad5 pIX-fusions such that that the added protein is elevated out of the crevices [9].

Here, four copies of Ad5 pIX are shown interlacing among four hexons (top and side views) (PDB 6B1T). The C-terminal domain α-helical bundle of pIX is clearly visible. The N-terminal domain triskelion structures are not visible in these views. Hexons are portrayed in cool colors and the pIX copies are shown in magenta. Some disordered regions are shown as dashed lines.

Protein IIIa

The Ad5 protein IIIa (pIIIa) plays a structural role in stabilizing the capsid from the inside [4]. Five copies of pIIIa are found under each vertex of the Ad5 capsid. It is 585 amino acids in length, but only residues 7 to 300 have been structurally traced at high resolution. Its N-terminal domain connects the penton and the five adjacent hexons. (These are known as the peripentonal hexons. The peripentonal hexons plus the penton are collectively named the group-of-six or GOS) Its C-terminal domain binds protein VIII (another structural protein which will be discussed later). The traced part of the pIIIa structure consists of two globular domains connected by a long α-helix.

Above, traced parts of five pIIIa proteins are shown on the underside of a part of the Ad5 capsid (perspective is from the interior) (PDB 6B1T). Hexons are colored blue, the penton is colored yellow, and pIIIa is colored bright pink. The same structure is shown below from a side perspective.

Protein VI

Ad5 protein VI (pVI) starts out as 250 amino acids long but is cleaved by AVP at two sites, yielding multiple peptides [4]. The first site is after residue 33 and the second is after residue 239. The middle part contains a predicted amphipathic α-helix (residues 34-54) which inserts into host endosomal membrane. This alters the membrane’s curvature and helps facilitate lysis of the endosome, allowing the adenovirus to escape into the cytosol. The middle part also contains a domain (residues 109-143) which sometimes binds to the inner surface of the capsid in the cavities between certain hexons. The N-terminal peptide pVI_N also binds to cavities between hexons. It has been suggested that this affinity hides the first pVI cleavage site in these cavities, preventing release of the membrane lytic peptide. During intracellular trafficking, environmental changes may allow adenovirus protein VII (a core protein) to outcompete pVI for the binding sites between hexons, causing release of the membrane lytic peptide. Finally, the C-terminal peptide pVI_C is a cofactor which helps activate AVP. The pVI_C peptide binds covalently to AVP and slides along the adenoviral genome, using the DNA as a track to reach all of the substrates in the core and the inner capsid surface. There are approximately 360 copies of protein VI in the Ad5 virion. Unfortunately, high-resolution structural data on pVI are scarce due to its variable position in the adenovirus virion.

Protein VIII

Ad5’s protein VIII (pVIII) also contributes to structurally stabilizing the adenoviral capsid from the interior [4]. It starts as a 227-residue protein which is cleaved by AVP at three sites, yielding two large peptides and two small peptides. The two large peptides stay together and bind between hexons. Some pVIII copies wedge between pIIIa and the peripentonal hexons, helping to connect the peripentonal hexons to the next set of surrounding hexons. Some pVIII copies are located underneath the nine hexons on the middle face of each icosahedral facet (known as the group-of-nine or GON). An interesting aspect of pVIII-hexon interactions is that can pVIII can engage in β-sheet augmentation, where a β-strand from pVIII is incorporated into one of the jellyroll domains of a nearby hexon. Not much is known about the two smaller peptides from pVIII except that these peptides do not appear to bind the capsid in a symmetric fashion.

Here, the traced parts of pVIII (red) are shown interwoven into a piece of the Ad5 capsid from an interior perspective (PDB 6B1T). Hexons are shown in shades of blue, the penton is shown in yellow, and pIIIa is displayed in bright pink.

Core proteins which interact directly with the capsid

Protein V

Adenovirus protein V (pV) is a positively charged protein which can form heterodimers with the pVII core protein [4]. That said, pV exists in a dimer-monomer equilibirium, so the binding to pVII is often transient. There also are direct associations between pV and the pVI capsid protein. These associations between pVII, pV, and pVI likely act to bridge the adenovirus core with the adenovirus capsid. In addition, pV-pVII heterodimers might interact with core protein μ. Each virion contains about 150 copies of pV. Most of the copies of pV are released during the beginning of uncoating. Interestingly, pV is not essential for adenovirus capsid assembly.

Protein VII

Protein VII (pVII) is a positively charged protein which plays a central role in condensing the adenovirus genome to fit into the capsid [4]. It has many arginine residues which contribute to its positive charge. AVP cleaves pVII at residues 13 and 24. The resulting middle peptide (including amino acids 13 through 24) might compete with pVI for hexon binding sites during adenovirus assembly. As mentioned earlier, environmental changes during intracellular trafficking may allow pVII to outcompete pVI for their hexon binding sites, causing release of the membrane lytic peptide from pVI cleavage. Though pVII acts as a functional analogue of the histone, it does not share much structural similarity with histones and does not replace histones when introduced into the cellular nucleus [2]. During infection, the viral genomic DNA as complexed with pVII is imported through nuclear pores. While pVII is important for condensing the adenoviral genome, it is not strictly required for assembly and packaging. In addition, pVII functions in signaling for the suppression of host innate immune responses. It binds to high mobility group B (HMGB) protein 1, a factor which is normally released from cells exposed to inflammation and which acts as a danger signal for the immune system. The adenoviral pVII prevents release of HMGB protein 1 and thereby dampens innate immune responses. Finally, pVII helps to regulate the progression of various steps during adenovirus genome replication.

Conclusion

This guide has centered on explaining the structures and functions of the Ad5 capsid proteins as well as the core proteins which are involved in key structural interactions with the capsid proteins. But this is only the beginning of learning about adenovirus biology. As mentioned in the introductory section, there are other core proteins including protein μ, the adenovirus proteinase, protein IVa2, and terminal protein which primarily interact with the adenovirus genome. Furthermore, the complex life cycle of the adenovirus requires numerous replication and packaging proteins (as well as interesting interactions with host cells) not covered here. Despite the specific focus of this guide, I hope that it is helpful to the reader for gaining a better idea of how the adenovirus capsid works. Perhaps this text will even provide a valuable bedrock of understanding for interested readers who are working on Ad5 capsid engineering projects.

References

[1] L. T. Collins and D. T. Curiel, “Synthetic Biology Approaches for Engineering Next-Generation Adenoviral Gene Therapies,” ACS Nano, Aug. 2021, doi: 10.1021/acsnano.1c04556.

[2] S. Kulanayake and S. K. Tikoo, “Adenovirus Core Proteins: Structure and Function,” Viruses , vol. 13, no. 3. 2021, doi: 10.3390/v13030388.

[3] Y. S. Ahi and S. K. Mittal, “Components of Adenovirus Genome Packaging,” Frontiers in Microbiology, vol. 7. p. 1503, 2016, [Online]. Available: https://www.frontiersin.org/article/10.3389/fmicb.2016.01503.

[4] J. Gallardo, M. Pérez-Illana, N. Martín-González, and C. San Martín, “Adenovirus Structure: What Is New?,” International Journal of Molecular Sciences , vol. 22, no. 10. 2021, doi: 10.3390/ijms22105240.

[5] W. C. Russell, “Adenoviruses: update on structure and function,” J. Gen. Virol., vol. 90, no. 1, pp. 1–20, 2009, doi: https://doi.org/10.1099/vir.0.003087-0.

[6] E. Vigne et al., “Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation,” Gene Ther., vol. 10, no. 2, pp. 153–162, 2003, doi: 10.1038/sj.gt.3301845.

[7] S. A. Nicklin, E. Wu, G. R. Nemerow, and A. H. Baker, “The influence of adenovirus fiber structure and function on vector development for gene therapy,” Mol. Ther., vol. 12, no. 3, pp. 384–393, Sep. 2005, doi: 10.1016/j.ymthe.2005.05.008.

[8] V. S. Reddy and G. R. Nemerow, “Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection,” Proc. Natl. Acad. Sci., vol. 111, no. 32, pp. 11715 LP – 11720, Aug. 2014, doi: 10.1073/pnas.1408462111.

[9] J. Vellinga et al., “Spacers Increase the Accessibility of Peptide Ligands Linked to the Carboxyl Terminus of Adenovirus Minor Capsid Protein IX,” J. Virol., vol. 78, no. 7, pp. 3470 LP – 3479, Apr. 2004, doi: 10.1128/JVI.78.7.3470-3479.2004.

Science Fiction Book Reviews

December 6, 2021January 11, 2026By logancollins 2 Comments

Spin by Robert Charles Wilson: 99/100. Science fiction is often said to be defined by a “sense of wonder”. I personally believe that the best science fiction features both a sense of wonder and a way of intertwining that sense of wonder with the deeply personal sense of what it means to be human. Spin does all this and more. It has relatable, complex, and believable characters who drive the story’s meaning. It gives us not one but several intensely awe-inspiring sci-fi concepts. It provides gorgeously philosophical and often poetic prose, a pleasure to absorb. In this book, the personal relationships of characters who grow up and change over the course of decades are pitted against a world experiencing a transformative crisis which forces the reader to consider powerful philosophical questions about what it means to live a good life as an individual as well as about humanity’s place in the universe. Though Spin spans a number of settings, cultural contexts, and technical areas (e.g. medicine, politics, aerospace, and more), the story is clearly very well-researched as it maintains an exceptional degree of verisimilitude. I won’t say much about the ending except that it is spectacular and even spiritually gratifying, though very much grounded in science. Spin is everything science fiction is meant to be. It gives us the incomparable gift of seeing life both at the level of human relationships and at the scale of the cosmos.

Solaris by Stanislaw Lem: 99/100. A remarkable feature of the human species is the desire to know, understand, and derive meaning. This can involve seeking to know oneself via introspection or seeking to know the universe via scientific inquiry. In Solaris, a mysterious alien entity which takes the form of a living ocean covering an entire planet acts as a centerpiece to examining questions of human knowledge, understanding, and purpose. As the crew of a research station orbiting Solaris begins to experience certain phenomena caused by the alien ocean, they are forced to confront their own pasts, traumas, and regrets. As a deeply emotional and psychological tale, Solaris examines the limits of human understanding both of the universe and of the self. While some ideas within the story may seem unsettling, I did not think it came across as an especially dark narrative, merely one which dispassionately portrays the human struggle for meaning against a backdrop of a strange and wonderous cosmos that often resists attempts to frame it within comprehensible human terms.

Station Eleven by Emily St. John Mandel: 99/100. Much of the essence of art is to reflect what makes us human, helping us better explain to ourselves what makes us tick. Station Eleven is a science fiction novel about a deadly flu pandemic which brings about the end of the world. Notably, it was written several years prior to the emergence of COVID-19. Emily St. John Mandel wields the premise masterfully to touch our souls and help us come to terms with human kindness, cruelty, hope, and vulnerability. Through its deep tragedy and heartfelt characters, the book manages to link questions of the individual and the global. We take a hard look at how the meaning of civilization connects to the meaning of life. Emily St. John Mandel’s prose puts billions to death. Those who survive must find purpose against the backdrop of the visceral viciousness of the apocalypse. Some immerse themselves in art, traveling the postapocalyptic wilderness and performing Shakespeare plays for pockets of survivors. Some join a religious cult led by a violent prophet who resembles history’s most monstrous men. Yet even this figure is skillfully humanized (though not exonerated) as having emerged from a frightened and damaged boy. Richly constructed character histories weave together in the end, creating a gorgeous tapestry which reveals both the inherent goodness and the intrinsic darkness of the human species. Station Eleven is lyrical, haunting, and intense. It immerses the reader in a realm which translates philosophy into the more brutally real language of emotion.

The Fall of Hyperion by Dan Simmons: 99/100. With its lyrical prose, immense cultural complexity, and epic scope, The Fall of Hyperion (hereafter referred to as “The Fall”) explosively propels into action the spring-loaded setup of the first Hyperion novel, kicking off a vividly imagined interstellar conflict of breathtaking scale and detail. I cannot overstate the awe-inspiring majesty and orchestral diversity of the Hegemony civilization as well as the incredible facility of Simmons with his extensive web of literary, religious, and historical allusions. Every line reads like poetry. The Fall possesses emotional depth to rival Dune and worldbuilding to compete with The Lord of the Rings. The Fall explores themes at the intersection of religious faith and penance, love and sacrifice, ecological destruction and the place of humanity in the universe, and (perhaps most centrally) artificial intelligence and God. Its history stretches both into the past and into the deep future with the Time Tombs sliding inexorably backwards in time as well as the ever-present specter of the terrifying Shrike and its Tree of Thorns having emerged from some distant tomorrow. After reading The Fall, I have taken on the conviction that a literary scholar could devote his or her entire career to studying the Hyperion tetralogy. The Fall is a mythopoetic elegy to humanity’s search for meaning in the cosmos, to the richness of our civilization’s vast story, and to our ultimate potential as a species.

This Is How You Lose the Time War by Amal El-Mohtar and Max Gladstone: 98/100. I have a special fondness for fiction which reads like poetry. This Is How You Lose the Time War by Amal El-Mohtar and Max Gladstone represents a tour de force of far-future poetic science fiction which sparkles with imagination, intensity, and wonder. An epistolary novel, it is told through letters exchanged by a pair of time-traveling cyborg supersoldiers named Red and Blue respectively who start as mortal enemies on opposite sides of a war and gradually fall in love. Each letter is delivered through a distinct medium; powdered cod bone sprinkled over a biscuit, a code of mineral veins in lava, a pattern of a bee’s flight and the venom of its sting, and many more. Red and Blue often spend decades in different pasts and futures, taking on the forms of various people and animals as part of their war. Though this conflict’s degree of convolutedness is far beyond human comprehension, the authors expertly utilize lyrical language to transmit a tantalizing taste of its scope. The central characters are so far beyond human that they should seem alien to the reader, yet their emotions come across as piercing and visceral. Beyond this, the beauty of the language gives the narrative a songlike quality which instills every passage with sensation, crispness, and vivacity. In terms of symbolism and metaphor, the book contains more than enough fractal complexity to fill the Library of Congress with multilayered literary analyses. This Is How You Lose The Time War furthermore incorporates a wealth of fascinating philosophical ideas involving love, war, peace, power, and freedom which are built on top of its spectacular wordsmithing. This book makes me feel like I am sipping liquid beauty during the cool of early morning while watching the stars of an alien sky slip beneath the horizon.

Blindsight by Peter Watts: 98/100. It is difficult to describe Blindsight. I could clumsily slap labels onto the novel and call it literary psychological sci-fi horror with an emphasis on the philosophy of neuroscience. I could vaguely refer to it as a boiling froth of darkness replete with nightmarish poetics. I could say that it manages incorporate both aliens and vampires in a terrifyingly believable fashion. I could pontificate on how the story oozes with malign hyperintelligence and conveys a sense of hurtling movement too fast to track with human eyes. Yet none of this can truly capture the frightening majesty of the narrative. More directly, Blindsight is a story about contact with aliens. After humanity first encounters the aliens, the governments of Earth send a group of cyborgs, freaks, and savants on a living spaceship to meet the aliens. The captain of this group is vampire, a technologically resurrected predator with intelligence vastly exceeding that of any human. The protagonist (Siri Keeton) had half his brain surgically removed when he was a child, rendering him incapable of empathy and forcing him to learn how to navigate social interactions through purely algorithmic techniques. Siri’s unusual backstory and motivations are richly explored over the course of the story. The novel explores ideas surrounding radical neurodivergence, transhumanism, the effects of neurotechnology on society, intelligence, consciousness, artificial intelligence, empathy, the blurring of the human-machine divide, emotional abuse, ableism, and evolutionary biology. As the book progresses, numerous psychological and philosophical revelations accrue. The aliens are more truly alien than any other aliens I have encountered in fiction. It is through a certain aspect of these aliens that the book’s most intensely frightening philosophical proposition is unveiled, but I will not spoil that for the reader. Prepare to be deeply disturbed in the most intellectually stimulating of ways.

The Chronoliths by Robert Charles Wilson: 97/100. Science fiction is the literature of ideas. Quality science fiction links these ideas to our own lives in a meaningful fashion. The Chronoliths by Robert Charles Wilson is a novel which successfully weaves together big ideas with intensely personal trajectories of individual human lives. Through this style of writing, it allows us to see ourselves in the characters and reflect upon our roles in the epic drama of civilization and the universe. The Chronoliths blends several stories into a unified narrative. It tells the story of icy monuments which periodically materialize at various locations across the Earth, causing death and destruction where they appear. These Chronoliths have writing on them, text which proclaims future military victories by a warlord named Kuin. It tells the story of an ordinary man named Scott Warden, his efforts to protect his daughter, and how his destiny is inextricably linked to the Chronoliths by the physical forces of nature. It tells the story of a genius physicist named Sulamith Chopra who finds herself increasingly obsessed with the Chronoliths and how they influence the flow of history. It tells the story of a single mother named Ashlee and her difficult relationship with her sociopathic son Adam Mills. I am struck by the deeply human identities of all of the characters (even many of the minor characters). They feel so vividly real with their struggles, quirks, backstories, and traumas. I tangibly feel their hopes and fears as they search for purpose in the midst of troubled world. All of this is accentuated by the lovingly detailed global setting which glows with verisimilitude. I should mention that I am a longtime fan of Robert Charles Wilson’s writings. His short piece Utriusque Cosmi is perhaps my favorite story of all time. Yet even with my high expectations going into The Chonoliths, I was nonetheless floored by its haunting beauty.

Hyperion by Dan Simmons: 97/100. Hyperion is no ordinary space opera. Its resplendent worldbuilding rarely follows traditional science fiction tropes, yet each and every careful detail of the galactic civilization called the Hegemony somehow feels right. The novel features exceptionally lush prose with blazingly colorful imagery and startling emotional revelations. It consists of six stories (modeled after the Canterbury Tales) told by characters on a pilgrimage across the planet Hyperion, interspersed by episodes describing their journey as they approach the domain of the Shrike, a spiky metallic entity that defies the laws of time and space. Each story reveals new insights about the Hegemony and the terrifying mystery of the Shrike. The stories include: (1) the priest’s tale and its examination of Christian faith in the context of the Shrike’s alien influence, (2) the soldier’s tale and its blend of sex and love and militarism and violence with the Shrike at its center, (3) the poet’s tale where an eccentric writer finds himself with the Shrike as his muse as he seeks truth through his poetry, (4) the scholar’s tale where a Jewish professor’s daughter contracts a strange illness that causes her to age in reverse after encountering the Shrike on a research expedition, (5) the detective’s tale where a tough-as-nails woman and a cybernetic recreation of the poet John Keats fall in love as they spar with the vast power of AI organization called the Technocore and grapple with its Ultimate Intelligence project, and (6) the consul’s tale featuring a touching tragic love story which takes place as an oceanic planet called Maui-Covenant is colonized by the Hegemony and the local population is forced to give up their cherished island culture. Hyperion concludes with a cliffhanger as the pilgrims arrive at the infamous Time Tombs and prepare to meet the Shrike. (The next book in the series is titled “The Fall of Hyperion”). In my view, this remarkably coherent blend of disparate narratives together forms a resplendent literary achievement. It will have a lasting influence on my own approach to science fiction writing and to thinking about the beauty of the world around me.

The Sparrow by Mary Doria Russell: 95/100. It is not easy to incorporate theology into science fiction without proselytizing the reader, yet The Sparrow does an elegant job of examining philosophy of religion through a first contact lens. At a deeper level, this book is about the human search for meaning and belonging in the universe, so even nonreligious readers can viscerally appreciate most of its ideas. Some other important themes the interplay between love, trauma, guilt, faith, anger, and healing. There are also some interesting (and reasonably balanced) forays into the psychology surrounding sexual abstinence of priests. The Sparrow charts the painful recovery of the sole survivor of a mission to make first contact with aliens through visiting them directly on their home planet. The survivor is Father Emilio Sandoz and he is physically disfigured and psychologically scarred by his experiences. The novel works backwards to explain what happened to him and the rest of the crew of the mission. This book includes some extremely disturbing occurrences. I believe that these occurrences were necessary for the story, but they might be triggering to some readers, so please be aware of this. On a lighter note, Mary Doria Russell’s writing clearly demonstrates her exceptional skills as a historian. Part of what makes this story feel so real is that it contains a wealth of impeccably researched cultural depth. Latin American settings, the history of Turkey, the bureaucracy of the Roman Catholic Church, and more are covered in loving detail. Furthermore, the characters show thoroughly believable backstories, quirky personalities, and complex psychological evolution. I care about these people. The Sparrow represents one of the most philosophically rich and thought-provoking books that I have yet encountered.

The Quantum Thief by Hannu Rajaniemi: 95/100. I would characterize The Quantum Thief as the most imaginative novel I have ever read. From beginning to end, it sparkles with kaleidoscopic strangeness. Though some readers might be put off by the onslaught of unfamiliar terminology, I found the bizarre language exhilarating. It tells the tale of a gentleman thief named Jean le Flambeur who goes through a series of convoluted adventures in a hyper-futuristic postsingularity version of our own solar system. The novel explores the unreliability of memory and mind in a future where advanced neurotechnology is ubiquitous and any dividing line between biology and technology has been completely obliterated. I possess great admiration for the sheer audacity of the Rajaniemi’s creativity. The walking city on Mars (called the Oubliette) where much of the story takes place is only the tip of the iceberg. When people die in that city, their minds are transferred into colossal robotic monsters known as the Quiet which toil beneath the city on the surface of Mars. A detective accesses the Oubliette’s exomemory to solve the mystery of a murdered Chocolatier. The living spaceship named Perhonen flirts with the thief protagonist. Every line of the book adds more of these kinds of concepts. As the plot cascades, complex mysteries of missing memories and buried pasts unravel. All this mixes with the thrill of the heist, a cast of believable and emotionally resonant characters, a complex alien political landscape, and a sense that this futuristic society has been oddly suffused with French culture. It is difficult to properly describe the profoundly colorful weirdness of The Quantum Thief. You just have to read it for yourself.

Holy Fire by Bruce Sterling: 95/100. While some may find Bruce Sterling’s Holy Fire meandering or indulgent, I found immense charm in its celebration of flamboyantly artsy cultural concepts. The novel explores the experiences of 94-year-old woman who undergoes a rejuvenation treatment to restore her biological age to around 20, then flees from the clinical authorities and from the USA to engage in a rebellious journey through a futuristic bohemian Europe. Replete with complex sociopolitical and scholarly aesthetic philosophy, the book is set against the backdrop of a world where gerontocratic governments have taken hold due to life extension treatments. Though many stories only show the negatives of longevity, Holy Fire examines the concept in a reasonably balanced manner and does not come across as polemical. It furthermore displays a strange yet mostly believable future with incredible attention to detail. I particularly appreciated the narrative’s deep exploration of creative subcultures such as those of fashion, photography, sculpting, and modeling as well as how such subcultures might manifest in a future with very long-lived people. Through its bohemian background, the book examines themes around the psychology of youth and old age, longevity technology, posthumanism, sexuality, political structures, and the meaning of artistic expression. Although the characters do not possess exceptional verisimilitude, their radically unique quirkiness manifests them in vivid color. Holy Fire draws strength more as a proverbial picture gallery of ideas than as a traditional written narrative.

Never Let Me Go by Kazuo Ishiguro: 95/100. For many, growing up is filled with both yearning and conflict. Never Let Me Go successfully captures the emotional intensity associated with the coming-of-age process while simultaneously investigating some dark concepts in bioethics. It is the story of Kathy, Tommy, Ruth, and a few others who grow up at an unusual English boarding school called Hailsham. The book chronicles the unfolding of their lives in a vividly believable and exquisitely detailed fashion as they hurtle towards an inevitable fate. They experience the familiar trials of growing up: navigating tricky social landscapes, falling in love, learning about the world, and forming their own identities. But there is a tragic context which overshadows these experiences. To reveal the specifics of this context would spoil some key aspects of the book, so I will only state that it explores some fascinating ideas in the area of medical science fiction. Despite the bioethics-related speculation which appears later in the novel, the narrative remains centered on the individual experiences of the characters, which fits well with its stylistic approach. Themes of mortality, love, friendship, and meaning are explored throughout. Perhaps most importantly, Never Let Me Go represents a deeply emotional story. By the end, I was weeping for the intricate characters who had decided to quietly accept something very sad indeed.

Exhalation by Ted Chiang: 95/100. As someone who was strongly influenced by Ted Chiang’s first short story collection “Stories of Your Life and Others”, I came into Exhalation with high expectations. I was not disappointed. Chiang possesses a special talent for crafting brilliant short pieces that combine intense clarity, tremendous conceptual ingenuity, and vast emotional depth. For instance, The Merchant and the Alchemist’s Gate followed the lyrical style of the classic One Thousand and One Nights, provided an uplifting narrative of loss and regret and redemption, and accessed themes of acceptance and fate. Another excellent story in the collection, The Truth of Fact The Truth of Feeling, gave a balanced perspective on how technology influences the way our brains think and communicate while also examining both a complex relationship between a father and daughter and a linguistics-driven historical scenario. The Lifecycle of Software Objects examines the concept of raising artificially intelligent creatures as children in a highly believable fashion. Exhalation (the title story) takes place in an alternate universe populated by a very different sort of life, yet it precisely interrogates ideas of vital importance to both the grand human condition and the deeply personal. Ted Chiang has once again demonstrated himself as one of the greatest short form science fiction authors ever to live.

The Three-Body Problem by Cixin Liu: 94/100. Reading this novel is a headfirst dive into a vividly imagined world where deep questions of human nature are confronted in the context of an impending alien invasion. Despite containing many highly fantastical elements, the Three-Body Problem possesses a clarity of thought that makes it highly believable. This is facilitated by how the Liu expertly interweaves the history of the Chinese Cultural Revolution into the early parts of the story. Another strength of Liu’s novel is how he demonstrates complex physical phenomena in a highly understandable and compelling fashion, leading to the intense sense of wonder that is at the core of the spirit of science fiction. At the larger scale, The Three-Body Problem explores how patterns of history repeat due to human psychology, helps us to consider the place of humans in the universe, and asks the open-ended question of whether humanity is a force of good or a force of evil. In my view, this intellectually exhilarating novel represents a titanic literary achievement and is quite deserving of the high level of recognition it has received.

Childhood’s End by Arthur C. Clarke: 92/100. It is not easy to capture the sheer sense of awe which comes from contemplating that which is beyond human comprehension. Childhood’s End delivers a shockingly provocative glimpse into the sublime while forcing the reader to contemplate the place of humanity in the universe. As humans, many of us enjoy telling ourselves stories about loving gods. Those inclined towards Lovecraftian tales take the opposite approach, conjuring up nightmares of cosmic monsters. Arthur C. Clarke unflinchingly finds a middle ground between these extremes. At the staggering conclusion of Childhood’s End, we experience both the cold realization of our own insignificance and a spiritually satisfying transcendence. Clarke proposes that to truly understand the divine, we may need to transform into something which is no longer even remotely human. Perhaps I am of the minority opinion that I am not repulsed by this notion, though I certainly do have some reservations about it. This is a spectacularly thought-provoking novel. My only complaint is that the first two sections of the book are significantly less compelling than its Earth-shattering conclusion, though they are necessary to set it up. Because the story was published in 1953, it includes some very outdated sexist assumptions and racist terminology. (As a person who has read some of Clarke’s later novels, I can attest that he improved over time in this regard). The characters and plot in the initial two-thirds of the book feel too stiff and detached for my taste. Nonetheless, this is more than made up for with the final portion of the story. If you want to think about the big questions and experience both extreme alienness and spiritual wonderment at the same time, you should read this book.

A Canticle for Leibowitz by Walter M. Miller Jr.: 75/100. Although A Canticle for Leibowitz is highly regarded by critics and does indeed present an extremely compelling broader idea, I found myself somewhat disappointed by how the story executed its central objectives. This is partially because I went in with exceptionally high expectations. I had hoped the story would represent a richly sweeping future history of humanity’s fall, hard-fought struggle to rise again, and tragic second fall which the novel is known for. Instead, I found a set of three fragmented narratives which only loosely connected to the overall thrust of the plot. The third section of the book did not (in my view) present a convincing case of why humanity was doomed to destroy itself a second time. Much of this section dwelt on tangentially-related religious philosophy which did not in my opinion possess sufficient universality to satisfy non-Christian readers. The second section of the book had some merits in the way it displayed the reemergence of basic and applied sciences yet suffered from a rather confusing and again tangential set of plotlines. I felt that the first section of the book was the most impressive, with well-developed characters, strong imagery, and an overall compelling setup of the challenges facing humanity after a nuclear apocalypse. I also felt the intersection of religious and secular concepts was much better handled and more balanced in this first part of the story. It believably showed how religion may shape a shattered world, it offered an intellectually stimulating account of how religious and secular impulses can work together, and it allowed readers to come to their own conclusions. Additionally, the characterization was much stronger in the story’s first section. Despite the narrative overall not meeting my perhaps unfairly high expectations, I feel that it did contribute meaningfully to my literary and philosophical learnings and I am glad I read it.

The Windup Girl by Paolo Bacigalupi: 70/100. As one of the darkest reflections on human nature I have encountered, The Windup Girl represents a truly dystopian novel. Set in a biopunk Bangkok, it centers around a future where climate change has led to severe resource limitations and most people struggle to survive. As a consequence, the worst is brought out in people. Most of the characters are selfish, greedy, cruel, or indifferent to the suffering of others. Oppression, starvation, and extreme violence are the norm. Western “calorie companies” control the world’s food supply. The character for which the book is titled is a windup girl, a genetically engineered slave who endures horrific sexual abuse after having been abandoned in Bangkok by her former Japanese master. Over the course of the story, themes of culture, colonialism, racism, resistance, Eastern religion, poverty, sexuality, power, and biotechnology are explored in depth. While I personally think the author’s vision is one of unrealistic pessimism, the story does present an interesting worst-case scenario that may be valuable to consider. Although none of the characters are ethical or likeable, many of them are complex and believable, inducing the reader to have sympathy for their plights. I would have rated this book much lower if not for its beautifully immersive prose, strong pacing, detailed cultural milieu, and excellently written (though mostly morally despicable) characters. As an extreme philosophical exercise, this novel delivers well. Nonetheless, it is highly violent and disturbing and thus potentially quite unpleasant to sensitive readers.

A Guide to CRISPR-Cas Nucleases

November 10, 2021November 25, 2021By logancollins 2 Comments

PDF version: A Guide to CRISPR-Cas Nucleases by Logan Thrasher Collins

Many different types of CRISPR-Cas nucleases possess biotechnological relevance. For a newcomer, the menagerie of Cas proteins may seem overwhelming. It can be challenging to decide which type of CRISPR system to employ in one’s research. To help address this issue, I compiled these notes. While my guide is certainly not comprehensive, it still covers a wide swath of important Cas proteins and may prove valuable as a starting point for those interested in getting a sense of the field. One should be aware that the field of CRISPR technology is moving rapidly, so some of the nucleases described here might eventually be superseded by newly discovered and/or newly engineered Cas proteins. I would also like to mention that since these notes are specifically focused on types of Cas proteins, I have omitted direct explanations of some important CRISPR technologies such as base editors, prime editors, and dead Cas systems. I also have not directly explained important CRISPR-related concepts such as non-homologous end joining (NHEJ), homology-directed repair (HDR), and adeno-associated virus (AAV) vectors. I encourage the reader to look elsewhere to learn about these subjects since they are vital for having a strong understanding of CRISPR biotechnology. I hope that you enjoy reading my notes and find them useful for your own scientific endeavors!

SpCas9

SpCas9 represents one of the first discovered and most commonly used CRISPR-Cas proteins.¹ It comes from Streptococcus pyogenes, a gram-positive bacterial pathogen. SpCas9 employs two nuclease domains to make blunt double-stranded cuts in DNA: the HNH domain for cutting the strand which pairs with the gRNA and the RuvC domain for cutting the other strand. The protospacer adjacent motif (PAM) of SpCas9 has the sequence 5’-NGG-3’, which limits the target sites that the nuclease can find. Though wild-type (WT) SpCas9 possesses a problematic level of off-target activity, several mutant variants of the enzyme have been engineered which give it much more precision.^2,3 As some examples, a few of these (but not all of them) include eSpCas9-HF, eSpCas9(1.1), and HypaCas9. The eSpCas9-HF and eSpCas9(1.1) enzymes maintain robust on-target cleavage while reducing off-target effects.³ The HypaCas9 enzyme has similar properties, but with even less off-target effects.²

SaCas9

At 1053 amino acids in length, SaCas9 is significantly smaller than SpCas9 (which is 1368 amino acids long).⁴ SaCas9 can be used in mammalian cells, employs NNGRRT PAM sites (R is A or G), and uses RuvC and HNH domains for cutting. But without further engineering, SaCas9 has lower target specificity even than SpCas9. Fortunately, mutant versions of SaCas9 which exhibit improved targeting accuracy have been developed. Tan et al. engineered SaCas9-HF, a version of the protein which has much less off-target activity relative to the WT SaCas9 and retains its on-target activity.⁴ With such improvements, SaCas9-HF can serve as a useful alternative to SpCas9.

LbCas12a

The LbCas12a enzyme makes staggered cuts using a single RuvC domain (and no HNH domain), uses T-rich PAM sites, and catalyzes its own crRNA maturation.⁵ LbCas12a comes from Lachnospiraceae bacterium ND2006. LbCas12a has another remarkable property: the binding and cleavage of target dsDNA activates a separate part of the protein which nonspecifically cleaves any ssDNA in its vicinity. This nonspecific trans-cleavage activity is thought to occur as a result of a conformational change in the LbCas12a protein which exposes its RuvC domain for broader ssDNA attack after binding to target dsDNA.⁶ It should be noted that other type-V Cas proteins including AsCas12a (see corresponding section), FnCas12a (from the bacterium Francisella novicida), and AaCas12b (from the bacterium Alicyclobacillus acidoterrestris) have been shown to exhibit the same capabilities.⁵ There furthermore exist many RNA-guided RNA-targeting Cas proteins which possess the same types of abilities.⁷ There are likely many other type-V Cas proteins with these capabilities as well. The activation of type-V Cas proteins to perform indiscriminate ssDNA cleavage after exposure to target dsDNA has been exploited as a target-induced signal amplification method to develop novel molecular diagnostics.⁶

AsCas12a

The AsCas12a protein (also called Cpf1) is derived from Acidaminococcus sp.,⁸ which are a group of anaerobic gram-negative bacteria. The protein exhibits several distinctive features compared to Cas9. AsCas12a utilizes a T-rich PAM site, unlike Cas9’s G-rich PAM. This is useful since it expands the possible targets for CRISPR. In particular, the T-rich PAM of AsCas12a can be useful when dealing with organisms that have AT-rich genomes such as Plasmodium falciparum. The naturally occurring form of AsCas12a does not require a tracrRNA, instead its CRISPR arrays are processed into just crRNAs, which serve to complete the functional AsCas12a-crRNA complex. Rather than creating blunt ends, AsCas12a makes staggered cuts with 4-5 nucleotide 5’ overhangs. This is useful since it increases the precision of non-homologous end joining (NHEJ) repair and allows insertion of DNA sequences at a chosen cut site with a desired orientation as specified by the base pairing of the insert with the overhang sequences. In addition, the AsCas12a protein employs a single RuvC domain to make its staggered cuts and does not have an HNH domain. AsCas12a has a lower tolerance for gRNA-target mismatches⁹ compared to SpCas9 and therefore demonstrates greater targeting specificity. As a result, AsCas12a shows fewer off-target effects overall. But it also has a lower editing efficiency compared to Cas9 proteins, which means that less cells receive any edits upon introduction of the AsCas12a. As described with LbCas12a, the AsCas12a protein also can carry out nonspecific ssDNA cleavage after it cuts to its target dsDNA.

AsCas12a ultra

WT AsCas12a possesses high targeting specificity, low off-target effects, and makes 5’ overhangs which facilitate correct insert orientation (see the section on AsCas12a). These properties represent desirable qualities for therapeutic gene editing, but the low editing efficiency of AsCas12a limits its therapeutic potential. Because of this, Zhang et al. (in a collaboration between Editas and Integrated DNA Technologies) developed an engineered version of the protein which was dubbed AsCas12a ultra.⁹ This AsCas12a ultra protein was created using directed evolution in bacteria. It has two point mutations relative to WT AsCas12a, M537R and F870L. These mutations grant the AsCas12a ultra extremely high editing efficiency while maintaining the protein’s low level of off-target effects. For a variety of target sites, Zhang et al. demonstrated nearly 100% editing efficiency in HSPCs, iPSCs, T cells, and NK cells using AsCas12a ultra. They also showed 93% efficiency for simultaneous disruption of three genes in T cells. When performing knock-in edits, Zhang et al. achieved efficiencies of 60% in T cells, 50% in NK cells, and 30% in HSPCs. These impressive numbers illustrate the utility of AsCas12a ultra as a broadly applicable tool for therapeutic gene editing.

AsCas12f1

The AsCas12f1 protein consists of only 422 amino acids, making it one of the smallest Cas proteins known.¹⁰ It comes from a type of gram-positive iron-oxidizing bacteria called Acidibacillus sulfuroxidans. AsCas12f1 makes staggered double-stranded breaks in target DNA and recognizes 5’ T-rich PAMs. Even with minimal engineering (just the construction of gRNA from combining its tracrRNA and mature crRNA), Wu et al. showed that AsCas12f1 exhibits usable levels of activity in mammalian cells.¹⁰ When expressed directly in mammalian cells via a plasmid, the protein achieved a maximum indel efficiency of 32.8%. When delivered to mammalian cells by AAV-DJ, the maximum indel efficiency was 11.5%. The AsCas12f1 protein possesses considerable promise as a compact therapeutic gene editing tool.

Kim et al.’s engineered Un1Cas12f

At 529 amino acids in length, the Un1Cas12f nuclease represents one of the smallest Cas proteins yet discovered.¹¹ This is useful since the small size of Un1Cas12f’s gene allows it to easily fit within AAV vectors. It comes from an uncultured archaeon and is classified as a type-V CRISPR nuclease, which utilize a C-terminal RuvC domain and do not possess an HNH domain. Though the original Un1Cas12f-gRNA complex has very low editing efficiency in eukaryotic cells, Kim et al. were able to intensively engineer the gRNA using a rational design strategy and achieve an 867-fold improvement of indel frequency in mammalian cells.¹² They also showed that the Un1Cas12f gene and gRNA gene could be delivered to the cells using AAVs. Because of its small size, Un1Cas12f may serve as an excellent scaffold for creating base editors and prime editors which fit inside of AAVs.

CasMINI

The CasMINI protein is another engineered CRISPR nuclease derived from Cas12f,¹³ which comes from an uncultured archaeon. This Cas12f is the same as the Un1Cas12f used by Kim et al.¹² Since Cas12f has little to no editing activity in mammalian cells, Xu et al. used rational design to optimize the associated gRNA and employed directed evolution to optimize the protein itself.¹³ CasMINI, a 529 amino acid protein, was the end result of these approaches. When CasMINI was modified to make dCasMINI-VPR (the VPR is a protein fusion which activates certain genes), it performed with comparable efficiency relative to the commonly used dLbCas12a-VPR. In some cases, dCasMINI-VPR actually outperformed dLbCas12a-VPR. When dCasMINI was modified by fusing base editor (ABE) domains at its N-terminus, the dCasMINI-ABE constructs performed base editing at comparable efficiency relative to dLbCas12a-ABE proteins. Because of their small sizes, the genes encoding the dCasMINI-ABE designs could easily fit into AAV vectors, though Xu et al. did not test this in their paper. Furthermore, even the genes encoding CasMINI prime editors should fit into AAV vectors. It should be noted that the most efficient dCasMINI-ABE base editing occurred in a narrow window precisely 3-4 bp downstream of the PAM site. When CasMINI was tested for its ability to perform gene editing by making indels, it showed significantly improved activity over Cas12f, though the editing efficiencies were still fairly low at around 5-10%.

Cas12j

The Cas12j enzyme, also known as CasΦ, comes from the genomes of huge bacteriophages of the Biggiephage clade.¹⁴ This is remarkable since CRISPR systems have usually been found in bacteria and archaea rather than viruses (though the prevalence of such machinery in viruses is perhaps underestimated). It has been hypothesized that Biggiephages use Cas12j to cut the DNA of other competing bacteriophages. There exist subtypes of Cas12j such as Cas12j-1, Cas12j-2, and Cas12j-3. All of the Cas12j nucleases are small at between 700 and 800 amino acids in length. The Cas12j nuclease cuts target dsDNA using a single C-terminal RuvC domain. Cas12j’s RuvC domain has a small amount of homology to the TnpB protein superfamily from which type-V Cas proteins evolved, yet it still shares <7% amino acid identity overall with type-V Cas proteins. Cas12j is most closely related to a type of TnpB group which is distinct from the type-V enzymes. The Cas12j nuclease catalyzes its own crRNA maturation using its RuvC domain (similar to the type-V nucleases). Unlike the type-V Cas proteins, Cas12j uses the same active site for both its RuvC cleavage of target DNA and its RuvC processing of the crRNA. It employs T-rich PAM sites which have fairly minimal target requirements. For example, the PAM of the Cas12j-2 subtype is 5’-TBN-3’ (B = G, T, or C). These minimal requirements give Cas12j expanded target recognition capabilities compared to other Cas proteins. Cas12j is active in vitro as well as within bacterial, human, and plant cells. Cas12j-2 (with a gRNA) has been observed to edit up to 33% of HEK293 cells. Though this may sound somewhat low, it represents an editing efficiency comparable to that initially reported for Cas9.

LwaCas13a

The LwaCas13a protein is a type-VI CRISPR nuclease and it cleaves RNA rather than DNA.¹⁵ It represents one of the most active types of RNA-guided RNA-targeting Cas proteins. LwaCas13a catalyzes the maturation of its own crRNA. The enzyme comes from Leptotrichia wadei, a type of anaerobic gram-negative bacteria found in saliva. LwaCas13a has demonstrated around 50%-80% knockdown of target RNAs in mammalian and plant cells. This is similar to the knockdown efficiencies of shRNAs, but LwaCas13a shows much lower off-target effects. When converted into dLwaCas13a, the protein can act as an RNA imaging tool. It has also been reported to have strong potential for therapeutics as well. One of the most important emerging applications of LwaCas13a (and similar Cas proteins) is that they can be used in diagnostics for infectious diseases.⁷ To do this, the LwaCas13a gRNA can be designed to target an RNA sequence from a desired pathogen. LwaCas13a can then be mixed with a short reporter RNA oligonucleotide which has a fluorophore at one end and a quencher at the other (the fluorophore is quenched by its close proximity to the quencher). If the target pathogen RNA is introduced, LwaCas13a will cleave said target RNA as well as activate nonspecific trans-cleavage activity (see section on LbCas12a), leading to cleavage of the reporter oligonucleotides. When the reporter oligonucleotides are cleaved, the fluorophore is released from the quencher, resulting in observable fluorescence. It should be noted that many CRISPR-based diagnostics require some form of target nucleic acid amplification step to increase signal prior to the usage of a Cas protein like LwaCas13a, though ways to mitigate this limitation are undergoing rapid development.¹⁶

Cas13bt

Kannan et al. identified Cas13bt1 and Cas13bt3 as useful RNA-targeting CRISPR nucleases since Cas13bt has some activity in human cells.¹⁷ Cas13bt1 and Cas13bt3 are small at just 804 amino acids and 775 amino acids respectively. It should be noted that Cas13bt also exhibits nonspecific nonspecific trans-cleavage activity (see section on LbCas12a) after cleaving its RNA target, which may allow its usage in diagnostics. Kannan et al. took advantage of the small sizes of Cas13bt1 and Cas13bt3 to develop compact RNA base editors. They fused an ADAR2 hyperactive adenosine deaminase catalytic domain onto dCas13bt1 and dCas13bt3. The resulting constructs were respectively named REPAIR.t1 and REPAIR.t3 and were shown to facilitate adenosine to inosine conversion in target RNAs. They also fused an ADAR2dd cytidine deaminase domain (which was itself created through directed evolution) onto dCas13bt1 and dCas13bt3. The resulting constructs were respectively named RESCUE.t1 and RESCUE.t3 and were shown to facilitate conversion of cytosine to uracil in target RNAs. Due to the small sizes of Cas13bt enzymes, all of these RNA base editors were small enough to fit inside of AAV vectors even alongside gRNA encoding sequences. The authors demonstrated successful AAV-mediated delivery to cells, but the editing efficiencies were low, so further optimization will likely be necessary.

CasX

The CasX nuclease represents a distinct type of Cas protein which does not share much sequence similarity with other types of CRISPR enzymes except for a RuvC domain.¹⁸ It is an RNA-guided DNA-targeting endonuclease which has minimal nonspecific trans-cleavage activity. Using its single RuvC domain, CasX creates staggered cuts (with about 10 nucleotide overhangs) in dsDNA complementary to its gRNA and adjacent to its TTCN PAM sites. CasX nucleases are <1000 amino acids in length, which is smaller than Cas9 and Cas12a. This could be useful for AAV-mediated delivery of CasX systems. There are different subtypes of CasX which come from different bacteria. Two of the known subtypes are DpbCasX (from Deltaproteobacteria) and PlmCasX (from Planctomycetes). DpbCasx can act in human cells, though it shows limited gene editing efficiency. PlmCasX generally has better efficiency at performing in human cells and can often achieve targeted disruption of genes in around a third of transfected cells. While this level of disruption is still modest, it is similar to the levels originally found with WT Cas9 enzymes before they were optimized for gene editing.

Un1Cas12f (previously known as Cas14a)

The Cas12f proteins represent a class of small CRISPR nucleases (400-700 amino acids in length) that are capable of RNA-guided cleavage of ssDNA or dsDNA depending on whether the gRNA or crRNA includes a PAM. They employ a RuvC domain for cleavage and do not possess an HNH domain. There are various subtypes of Cas12f, but Un1Cas12f (previously Cas14a1) has been studied in the most detail. Un1Cas12f was first reported to selectively cleave ssDNA and not dsDNA.¹⁹ It was also initially reported to not require a PAM site for targeting. Without the constraint of needing a PAM site for targeting, Un1Cas12f has broader possibilities for which ssDNA sequences can be targeted. However, later research revealed that Un1Cas12f can cleave dsDNA when a 5’ T-rich PAM sequence is included in the gRNA or crRNA.²⁰ As with many other types of Cas proteins, Un1Cas12f exhibits nonspecific nonspecific trans-cleavage activity of dsDNA (see section on LbCas12a) after cleaving its target DNA, which grants it utility as a component of diagnostics.

DiCas7-11

The Cas7-11 protein is an RNA-guided RNA-targeting CRISPR nuclease.²¹ It is named Cas7-11 because it arose evolutionarily from a fusion of a protein known as Cas7 with a protein known as Cas11. The DiCas7-11 enzyme comes from the gram-negative sulfate-reducing bacteria Desulfonema ishimotonii (there also exist similar types of Cas7-11 from other species). An important advantage of DiCas7-11 is that it does not have a toxic effect on host cells (bacterial or mammalian). By comparison, RNA knockdown technologies including shRNA, LwaCas13a, PspCas13b, and RfxCas13d typically cause around 30-50% host cell death. DiCas7-11 shows similar knockdown efficiencies compared to these other RNA knockdown technologies while demonstrating no detectable cellular toxicity. Unfortunately, DiCas7-11 is also fairly large at 1602 amino acids, making it difficult to package into AAV vectors. One more application of Cas7-11 is RNA editing. The creation of a dDiCas7-11 fused to a base editor domain has enabled RNA editing in mammalian cells.

References:

3D structure images were created using PyMol.

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Resource: List of Biotechnology Companies to Watch

May 10, 2021November 17, 2025By logancollins 5 Comments

PDF version: List of Biotechnology Companies to Watch – by Logan Thrasher Collins

I created this list of organizations (162 total to date) to serve as a resource to help people learn about and keep track of key biotechnology companies. Some of these are emerging startups, some are established giants, and some provide useful services. Some notable nonprofit research organizations are included as well. Though this list is far from comprehensive, I have tried to cover as many of the key players as possible. It is also important to realize that this landscape is constantly changing, so some of the information on this list will eventually transition into antiquity. The list was originally started over the course of 2021, updated during the summer of 2022, updated during the summer of 2024, updated in January 2025, and updated in September-October 2025. I hope you enjoy delving into the exciting world of biotechnology!

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Company	Category	Description and Key Facts
Ablynx	Services	Nanobodies as therapeutics and as laboratory reagents.
Aera Therapeutics	Biomed	Developing protein nanoparticle delivery vehicles (originally the “selective endogenous encapsidation for cellular delivery” or SEND platform) for gene therapy which are based on proteins from endogenous virus-like particles encoded by the human genome. Also developing proprietary gene editing proteins of compact size to overcome packaging limits. Co-founded by Feng Zhang. Raised $193M in a February 2023 funding round.
AgeX Therapeutics	Biomed	Treating aging using stem cell therapies, induced tissue regeneration, related methods.
Aldevron	Services	Provides manufacturing and development services such as large-scale plasmid DNA synthesis, mRNA production, gene editing production, and antibody manufacturing. In 2025, worked with Integrated DNA Technologies to manufacture a personalized (N of 1) gene editing therapy for an infant with (otherwise untreatable) urea cycle disorder consisting of a lipid nanoparticle, custom gRNA, and an mRNA encoding a base editor. Manufacturing took 6 months, three times faster than the standard timeline. As of May 2025, the treatment was successful.
Allonnia	Ecotech	Engineering microorganisms and enzymes to degrade environmental pollutants. Funded by the Ferment Consortium of Ginkgo Bioworks.
Alora	Ecotech	Engineering salt-tolerant rice via CRISPR for ocean agriculture to feed the world. Formerly known as Agrisea. Early stage: raised a $1.4M seed round as of September 2022.
Altos Labs	Biomed	Developing cellular rejuvenation technologies to reverse age-related diseases and aging. Has raised over $3B from funders such as Jeff Bezos, Yuri Milner, and others (the most funding of any biotechnology company as of June 2024). Steve Horvath is one of the principal investigators working at Altos Labs. Main scientific advisor is Nobel Laureate Shinya Yamanaka.
Apertura Therapeutics	Biomed	Has developed TfR1-binding AAV capsids which efficiently cross the blood-brain-barrier while avoiding excessive liver transduction. Based on research published in Science by Huang et al. wherein mice expressing humanized TfR1 were used. Has engineered CD59-binding AAV which can cross the blood-brain-barrier, target brain tissue, and target muscle tissue. Founded by Ben Deverman, director of vector engineering at the Broad Institute. Leveraging cutting-edge machine learning tools to engineer AAV capsids simultaneously optimized for immune evasion, manufacturability, and tissue targeting. Collaborating with Dr. Sonia Vallabh at the Broad Institute to support her efforts to cure the prion disease fatal familial insomnia (which she herself will probably develop due to family genetics). Announced licensing deal with the Rett Syndrome Research Trust (RSRT) towards developing AAV-based treatments for Rett syndrome. Announced licensing deals with Galibra Neuroscience (for treating GABA-imbalance disorders) and Emugen Therapeutics (for treating neurodegenerative disorders) in August 2025. Launched with a $67M series A during 2022.
Aptah Biosciences	Biomed	Developing a rationally designed single-stranded DNA (lead compound APT20TTMG) that crosses the blood-brain-barrier, restores cellular RNA integrity, and corrects multiple proteins to facilitate brain rejuvenation and combat aging. APT20TTMG binds to pre-mRNA and facilitates proper U1 snRNP assembly to globally decrease cleavage of pre-mRNAs, decrease abnormal RNA splicing, decrease expression of inappropriately truncated proteins, and restore functionality of regulatory miRNAs. George Church and Aubrey de Grey are on the scientific advisory board.
Arena Bioworks	Biomed	Ended its operations as of November 2025 and is now defunct. Not a company but a biomedical research institute that employs principal investigators to lead basic research into the mechanisms of human disease, to develop therapies, and then to create spinoff companies that can translate those therapies to the clinic. Emphasizes translation by providing its investigators with the support and infrastructure to do so. Relies solely on private funding, thus its investigators do not need to apply for grants and can focus on the research. CRISPR pioneer Keith Joung is one of the first principal investigators at Arena. Co-founder and CEO is Stuart Schreiber, who also co-founded the Broad Institute. Launched with $500M in private funding. Located in Cambridge near MIT and Harvard.
Asimov	Biotech	Developing computer aided design tools for synthetic biology, making host cell lines for viral vector and biologics manufacturing, constructing genetic parts database. One of the co-founders is Christopher Voigt. James Collins is on the scientific advisory board.
Atomic AI	Bio-AI	Has developed AI tools for RNA 3D structure prediction as well as wet lab assays for evaluation. Their large language model ATOM-1 uses chemical mapping data to improve RNA optimization, predicting structural and functional properties of RNAs. Their foundational RNA structural prediction technology was published in Science during 2021. Leveraging their tools to develop therapeutic RNA-targeted small molecules and RNA-based medicines such as mRNA vaccines, siRNAs, and circular RNAs.
Aukera	Biomed	Developing protein vault delivery vectors for delivery of peptides, small molecules, and possibly nucleic acids. Their vault formulation is reportedly stable for years at room temperature.
Beam Therapeutics	Biomed	Developing base editor technologies towards therapeutic applications. David Liu and Feng Zhang are among the co-founders.
Bexorg	Neurotech	Has developed instrumentation for maintaining the cellular functions (but not the consciousness) of whole human brains from deceased donors. Their machinery perfuses the brains with an artificial blood-like substance and continuously measures substances going into and out of the brains. This allows automatic adjustment of oxygen and nutrient levels as well as measurement of the responses of the brains to potential therapies. To prevent consciousness, the brains are kept in a low-energy state where the neurons do not exhibit electrical activity. As an additional measure of caution, anesthetics are included in the perfusate. They collect -omics data from experiments on the brains, using these data to create a map of how the brains respond to potential therapeutics over time. Their data are fed into AI software to create predictive models for how therapies interact with human brains. Aims to improve success rates in CNS clinical trials through their data and models of human brain responses. Spun out from Nenad Sestan’s laboratory at Yale University. Has raised $42.5M as of October 2025.
BigHat Biosciences	Bio-AI	Has developed a high-speed closed-loop pipeline (known as Milliner) for antibody discovery, development, and optimization which leverages wet lab automation, advanced machine learning techniques, and synthetic biology. Milliner starts with preexisting antibodies, phage display, or generative AI to discover initial hits. It then rapidly produces antibodies and comprehensively characterizes them to obtain training data, before updating them with AI models that optimize the design. This cycle repeats in a loop until high-quality antibodies with desired characteristics are created. Also develops AI-optimized VHHs, camelids, BiTEs, scFvs, and fusions of different antibody-related components. In 2022, made strategic partnerships with Merck and Amgen as well as acquired a cell-free protein synthesis company called Frugi Biotechnology. Has several preclinical therapeutic programs in development (one of which is at IND-enabling stage) as of September 2025. Has raised over $100M in total as of September 2025.
Bioasis	Biomed	Has developed a peptide called xB³ that facilitates transcytosis across the blood-brain barrier. Working towards applications in glioblastomas, brain metastases, and neurodegenerative diseases.
Biogen	Biomed	Large pharmaceutical company focusing on developing treatments for neurological diseases. Has made moves towards developing gene therapy pipelines for treating neurological diseases, though the company has experienced some setbacks in this space (i.e. failed clinical trials).
BioMarin Pharmaceutical	Biomed	Enzyme replacement therapies for rare diseases. During April 2021, announced a collaboration with the Allen Institute to develop AAV gene therapies for rare diseases of the brain.
Bionaut Labs	Biomed	Microrobotics as a new paradigm for drug delivery.
BioViva	Biomed	Developing gene therapies to treat aging, offers tests for determining biological age. Elizabeth Parrish (the company’s CEO) tested an experimental gene therapy on herself and reports positive results, though she did not intend for this information to go public. George Church and Aubrey de Grey are on the scientific advisory board. Anders Sandberg is the company’s ethics advisor.
Blackrock Neurotech	Neurotech	Owns commercial rights to the Utah Array, one of the best known and most widely used neural electrode array technologies. The Utah Array was first implanted in humans during 2004 and has been used in clinical studies since then. Licenses the Utah Array to academics, companies, and clinicians. Developing brain-computer interfaces for control of prosthetic limbs, control of computer functions, writing text via computer, and restoration of senses (touch, vision, and hearing). Sells devices for human studies, non-human primate research, and rodent research. Has restored sensory or motor function to over 40 human patients through their studies. Their devices have remained functioning in patients for a total of 30,000+ days (adding up how long in each patient). In 2021, received FDA Breakthrough Device Designation for their MoveAgain medical device, which facilitates control of cursors and keyboards, mobile devices, wheelchairs and prosthetic devices. During 2024, received a large infusion of funding in the form of $200M worth of cryptocurrency cash (before that, they had only taken on about $10M in funding despite their longevity as a company). Is not at all affiliated with the well-known finance company Blackrock investments.
Calico Life Sciences	Biomed	A subsidiary of Alphabet Inc. (Google) which focuses on studying and treating aging. Partnered with Abbvie to develop drugs for age-related diseases. Has also established partnerships with the Broad Institute of MIT and Harvard and with the Buck Institute for Research on Aging, has published numerous peer-reviewed papers on the biology of aging.
Capsida Biotherapeutics	Biomed	Developing targeted AAV gene therapies for a variety of brain diseases. Has made blood-brain barrier crossing AAVs that are liver untargeted and brain targeted. Founded by Viviana Gradinaru.
Capsigen	Biomed	Engineering superior AAV gene therapy vectors through a proprietary method called Transcription-Dependent Directed Evolution (TRADE^TM). Have developed greatly improved neurotrophic AAVs. Entered into a partnership with Biogen during May of 2021 to develop AAV gene therapies that treat various brain and neuromuscular disorders.
Caribou Biosciences	Biomed	Developing allogenic CAR-T and CAR-NK therapies using a Cas12a chRDNA (CRISPR hybrid RNA-DNA) genome-editing technology which enables multiplex gene edits, higher specificity, and less off-target editing. As of June 2023, has two CAR-T therapies for hematologic diseases in phase I clinical trials as well as a portfolio of other therapies at earlier stages of development. Jennifer Doudna is a co-founder and is on the scientific advisory board. As of June 2023, has raised $167.7M in funding.
Cathedral Therapeutics	Biomed	Encapsulating AAVs inside of protein vaults as a way of shielding from preexisting anti-AAV immunity found in up to 60% of patients, a platform technology to increase access to gene therapy and improve the efficacy of genetic treatments. Based on early data, vaultAAVs might also enhance AAV transduction efficiency by about 5-fold, which could enable treatments at lower doses and thus make AAV gene therapy safer and less expensive. My company, which I co-founded with David Curiel.
CATALOG	Biotech	Building a DNA-based platform for massive digital data storage and computation.
Celero Systems	Biomed	Developing ingestible pills which can diagnose, monitor, and treat diseases by sending data to external devices (e.g. cell phones). Their HEALTH-Dx^TM pill can monitor respiratory and cardiac rhythms to diagnose sleep apnea. Their RESCUE-Rx^TM pill can automatically administer rescue medication in the case of an opioid overdose. Robert Langer is an advisor and one of the co-founders.
Cirsium Biosciences	Biomanufacturing	Developing a platform for manufacturing clinical-grade AAVs at higher yields which uses plants. They deliver plant-specific helper plasmids into producer plants using a vector (possibly A. tumefaciens), produce the AAVs inside the plant tissues, and purify the AAVs. Have demonstrated 70% reduction in manufacturing lead times compared to traditional methods. Produces up to 1015 vg/kg AAVs and is highly scalable, lowering costs compared to traditional methods. Safer than traditional methods since they leverage plants designed for resistance to contamination with human pathogens. Received (up to) $61M in ARPA-H funding as of October 2024.
Coastal Carbon	Ecotech	Aggregating massive amounts of satellite imagery data as well as non-intrusive underwater sensors to train foundation models and measure seaweed biomass, facilitating access to blue carbon for seaweed farmers. This strategy could accelerate the development of seaweed farming towards carbon capture. As of September 2023, raised $1.6M to develop the non-intrusive underwater sensors that can capture data to enhance the accuracy of their models.
Code Biotherapeutics	Biomed	Has developed 3DNA, a multivalent DNA nanostructure (not DNA origami) which both carries therapeutic transgenes and can be linked to antibodies or peptides to facilitate cell-targeted delivery of said transgenes. Focusing on Duchenne Muscular Dystrophy while also in very early stages of exploring lung, pancreas, and liver diseases. Has raised $85M as of June 2023.
Cognito Therapeutics	Neurotech	Developing a noninvasive wearable visual and audio stimulation device to evoke gamma waves in the brain, slowing cognitive decline of Alzheimer’s patients. Based on studies from Ed Boyden’s and Li-Huei Tsai’s labs at MIT. Co-founded by Ed Boyden and Li-Huei Tsai. As of September 2024, has completed a phase II clinical trial and demonstrated up to 77% reduction in cognitive decline over a period of 6 months with patients using the device.
Cognigenics	Biomed	Developing inhalable AAVs to deliver CRISPR gene therapy for treating anxiety, depression, and mental impairment. Has demonstrated successes in mouse models for treating anxiety as of June 2023. Plans to start clinical trials in 2024 and claims that they may bring the product to market as early as 2025. Leveraging contract research organizations (CROs) and contract manufacturing organizations (CMOs) to accelerate their research and development. First raised initial funding in 2020 for early preclinical work from early angel investors and then received $950K during 2022 from Fifth Set Ventures and Lionheart Ventures for further preclinical studies and beyond.
Constellation Bio	Biomed	Developing molecularly precise probiotics that actually work. First target is to develop precision medicine probiotics for lowering cholesterol. Pre-seed stage as of October 2025, funded by angel investors. As of October 2025, the company is collecting fecal samples along with some data about cardiovascular health from volunteers as part of their R&D. Leveraging observational human research to bypass much of the red tape often associated with early-stage therapeutic products. Founded by Stephen Skolnick, who announced the company in a Substack post.
Colossal	Ecotech	Centered on moonshot projects that are using advanced CRISPR methods to bring back the Wooly Mammoth, the Thylacine (Tazmanian Tiger), and other extinct animals. Aims to reintroduce lost biodiversity and thus repair ecosystems. Will develop biomedical technologies such as artificial wombs in conjunction with its de-extinction research, providing additional benefits to humanity and acting as a way to bring in funding. Cofounded by George Church, Ben Lamm, and Andrew Busey. As of January 2025, received a $200M series C funding round (for a total of $435M funds raised) at a $10.2B valuation.
Composite Programmable Therapeutics	Biomed	Has developed DNA origami shells to multivalently capture viruses and trigger their clearance by the immune system. Co-founded by Hendrik Dietz. As of June 2023, also planning to develop new gene therapy vectors based on DNA origami as well as a biomanufacturing platform for producing large quantities of ssDNA. Has raised $29M as of September 2024.
Concerto Biosciences	Biotech	Has developed a high-throughput assay device (kChip) which displays millions of microbial communities with different combinations of microorganisms. Across these microbial communities, pathogen suppression, metabolite production or degradation, robustness to environment, and other metrics are tracked. Data from kChip assays are used to train kAI to predict microbial behavior in different community contexts, which allows identification of combinations of microorganisms that work together to achieve useful functions. They are leveraging kChip and kAI to develop multistrain probiotics for dermatology applications including treatment of vaginal yeast infections, general skin health, and treatment of atopic dermatitis. As of this writing (October 2025), a phase 1b clinical trial has been completed for the probiotic formulation aimed at treating atopic dermatitis. Has raised $25.5M as of October 2025.
Convergent Research	Services	Not a company but a nonprofit organization which incubates, finds philanthropic donors for, and supports Focused Research Organizations (FROs). For more information on FROs, see this open access article in Nature. Adam Marblestone is CEO and a co-founder.
Cortical Labs	Neurotech	Developing hybrid bioelectronic devices which incorporate cultured biological neurons to perform computational tasks. These devices are power efficient, scalable, robust to physical damage, and have the potential for fluid adaptation to many different computational problems.
Cradle	Neurotech	Aiming to develop reversible whole-body cryopreservation for humans. They have so far shown that electrophysiological activity can be restored in a cryopreserved and rewarmed slice of rat cerebellar tissue. Has raised $48M as of June 2024. Laura Deming and Hunter Davis are the co-founders.
Creative Biolabs	Services	Custom services for antibody engineering, membrane protein production and characterization, bioconjugation, gene therapy development, viral vector engineering, cell therapy development, molecular dynamics simulations, drug development consulting, and more.
Cultivarium	Biotech	Developing molecular techniques, hardware platforms, and software tools to accelerate adoption of non-model microorganisms for biotechnology. Cultivarium is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Dalan Animal Health	Ecotech	Has developed the world’s first bee vaccine (made using inactivated bacteria), which protects hives against American Foulbrood disease, a devastating infection caused by the bacterium Paenibacillus larvae that spreads rapidly and leaves persistent spores. Sells their vaccine for beekeepers to mix with queen candy or in the form of vaccinated queens. American Foulbrood disease harms ecosystems and costs the U.S. food industry over $400M in lost revenue. Antibiotics used to control American Foulbrood disease can lead to resistance and can negatively affect the health of the bees; the vaccine does not lead to antibiotic resistance or harm the bees. Also developing a number of other vaccines for various honeybee infectious diseases (e.g. a vaccine against deformed wing virus is in clinical development as of December 2024) as well as one for diseases in shrimp. Raised about $10M in total funding as of June 2023.
Deep	Ecotech	Developing modular undersea habitats that scientists will live inside of for extended periods of time during marine research missions. Also offers advanced manufacturing services for customers seeking large metal parts. Funded with £100M+ by a mysterious anonymous donor (as of September 2025). UK-based company.
DoriVac	Biomed	Developing DNA origami cancer vaccines which facilitate cross presentation of antigens and are also applicable to infectious diseases. Raised $100K by winning the 2022 Alnylam BioVenture Challenge. Early stage as of June 2023. Their technology was developed by William Shih and Yang Zeng at the Wyss Institute.
Dyno Therapeutics	Biomed	Using deep learning to improve properties of AAV capsids as a platform technology for gene therapy. George Church is one of the co-founders.
E11 Bio	Neurotech	Building moonshot technologies involving superior molecular barcoding, spatial -omics, and viral circuit tracing to help neuroscientists map the brain. Has a long-term goal of mapping brains at the one-hundred billion neuron scale. E11 Bio is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Editas Medicine	Biomed	CRISPR-based gene therapy. George Church, David Liu, Jennifer Doudna, Feng Zhang, and J. Keith Joung are the co-founders.
eGenesis	Biomed	Developing safer xenotransplants by using multiplexed CRISPR gene editing to inactivate all of the porcine endogenous retroviruses and to address the numerous mechanisms of immune-mediated rejection. Working on gene edited porcine kidneys, livers, and hearts for xenotransplantation. George Church is a co-founder. Has raised a total of $481M in funding as of September 2024.
Eikon Therapeutics	Biomed	Superior drug discovery platform which leverages high-throughput automated super-resolution microscopy for tracking single protein movements in living cells. Eric Betzig is one of the advisors.
Emerald Cloud Lab	Services	Remote automated laboratory as a service for researchers. Has a large array of automated equipment for synthetic biology and genetic engineering, physical and biophysical chemistry, structural biology, biochemistry, analytical chemistry, etc. Provides a software interface for users to instruct the automated equipment.
Entos	Biomed	Developing lipid nanoparticles with transmembrane fusogenic proteins to facilitate delivery of DNA, RNA, and CRISPR cargos. Neutral lipid formulation (not ionizable) gives lower toxicity while the fusogenic proteins facilitate delivery efficacy. As of June 2024, Entos is involved in oncology therapeutics, antivirals, gene editing therapies, immunotherapies, DNA vaccines, and senolytics. Has partnered with Oisin Biotechnologies (see later in this list) to develop a new senolytic therapy.
Eve Bio	Biomed	Mapping the “pharmome” with the goal of identifying all of the off-target effects of clinically approved small-molecule drugs. Leveraging high-throughput screening assays with 2,000 FDA-approved drugs on reporter cells, measuring the effects on up to 1,000 gene products per drug. Many of their assays use reporter cell lines engineered to emit a quantifiable optical signal when a test drug stimulates a specific gene product (a different cell line for each gene product). Releasing their data and assay designs to the public to support pharmacological safety profiling, drug repurposing, biomedical AI training, polypharmacology (finding drugs that act at multiple targets), chemical toxicology profiling, and future automated laboratory processes. Eve Bio is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
EvolutionaryScale	Bio-AI	Has developed an open-source AI frontier model for biology (called ESM3) which can create a wide variety of proteins with desired functions through natural language prompting. ESM3 can create new proteins that diverge greatly from naturally occurring proteins including a variant of GFP with only 58% sequence similarity to the closest known naturally occurring type of GFP (calculated as the equivalent of simulating 500 million years of evolution). Has plans to develop plastic-degrading and carbon capture proteins using their models. An enormous amount of compute was used to train ESM3, it has 98 billion parameters, and it can be fine tuned by experimental results in a fashion analogous to RLHF (reinforcement learning with human feedback). ESM3 can reason across protein sequence, structure, and function, making it extremely generalizable. Even more compute resources and data for training could create new models with even greater generative capabilities and new models that operate across biological scales from individual molecules to whole cells. Patrick Hsu is one of their investors and is listed as an author on the ESM3 paper in the journal Science. Led by CEO Alexander Rives, former head of Meta’s AI protein team. Raised a seed round of $142M as of June 2024.
Evox Therapeutics	Biomed	Developing exosomes loaded with AAV as a delivery system for gene therapy, shielding AAVs from immune factors and targeting them to specific tissues. Also exploring other exosome cargos such as RNAs, CRISPR-Cas proteins, and therapeutic proteins. Preclinical stage as of June 2023.
Fauna Bio	Biomed	Has developed a platform called Convergence^TM AI which uses data from the Zoonomia Consortium, including genome alignments and protein-coding alignments of hundreds of mammalian species. Convergence^TM AI identifies protective genomic signatures in disease-resilient mammals such as hibernators (e.g. 13-lined ground squirrels) and maps these results to human cell models so that validation experiments can be performed. It also predicts compounds which can mimic the protective genomic phenotypes upon application to human cells. Working on preclinical development of drugs for cardiopulmonary and retinal indications as of October 2025. In January 2020, announced a collaboration with Novo Nordisk to discover new treatments for obesity. In December 2023, announced a partnership with Eli Lilly for preclinical drug discovery in obesity. This partnership renders Fauna Bio eligible to receive up to $494 million in preclinical, clinical, and commercial milestone payments as well as royalties on potential product sales. In addition to past funding, has raised a $40M series A as of March 2025.
Fieldstone Bio	Ecotech	Developing microbial biosensors which synthesize hyperspectral reporter molecules through engineered metabolism in response to desired signals on the ground (e.g. contaminants in soil). The hyperspectral reporters can be imaged from above using flying drones equipped with special cameras, enabling rapid surveying of chemical properties across large amounts of land. Applying AI software to automate analysis of images taken by drones. Application areas include contaminant detection, agriculture, national security, and mining. Their foundational technology is based on published research by Chemla and Levin et al. from Christopher Voigt’s laboratory at MIT.
Forest Neurotech	Neurotech	Developing a minimally invasive ultrasonic brain-computer interface implant that can access any part of the brain to understand and treat a wide range of neurological disorders. Will employ both ultrasonic neuroimaging and neuromodulation using Ultrasound-on-Chip technology from their partner Butterfly Network. Sumner Norman is CEO and a co-founder. Launched in 2023 with $14M in philanthropic funding from Convergent Research, has since raised additional funding. Has signed $20M contract to pay Butterfly Network for facilitating partnership and licensing of the Ultrasound-on-Chip technology. Forest Neurotech is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Form Bio	Services	Developing AI-powered computational services for characterization and prediction of the properties of engineered AAVs (e.g. simulation and analysis of bioreactor setups for AAV production, prediction of mRNA expression, immunotoxicity prediction, generative in silico AAV candidate optimization) as well as for analyzing data from AAV production. Spun off by another startup company (see earlier in this list) called Colossal. Some of its advisors include George Church, Christopher Mason, and Peter Diamandis.
Frontier Bio	Biotech	Has developed tissue engineered systems including Blood Vessel Mimics (already in use for medical device testing, a neurovascular-unit-on-a-chip for studying the blood-brain-barrier in health and disease, and vascularized organoids for aiding drug development and disease modeling. Has raised $1.1M from investors and an SBIR grant as of July 2023.
Future House	Bio-AI	Not a company but a research nonprofit organization funded through philanthropist Eric Schmidt, also seeking other funding. They plan to spend $20M ramping up during 2024. Has a 10-year mission to create “AI scientists”, semiautonomous AI systems that may dramatically accelerate the pace of biological research through not only laboratory automation, but also through cognitive automation of literature research, protocol writing, generating hypotheses, discerning patterns in data, etc. Founder and CEO is Sam Rodriques, a principal investigator at the Crick Institute.
Gameto	Biomed	Developing women’s reproductive health technologies, starting with Fertilo. Fertilo consists of lines of ovarian support cells that secrete hormones to mature eggs in a dish, replacing hormonal injections and shortening the IVF process from 14 days to 3 days. Eggs can be frozen or fertilized after Fertilo is used. Raised $73M in total funding as of December 2024. In December 2024, announced birth of first human baby who came from eggs matured using the Fertilo technology.
GATTAquant	Services	DNA origami imaging probes, fluorescence microscopy reagents. First commercial application of DNA origami.
Generate Biomedicines	Bio-AI	Generative artificial intelligence to create novel de novo protein therapeutics with desired protein-protein interactions, enhanced enzymatic activities, and invisibility to the immune system. Frances Arnold is on the board of directors. Has raised $420M as of July 2023.
Generation Bio	Biomed	Developing gene therapies for rare and prevalent genetic diseases using close-ended DNA and cell-targeted lipid nanoparticle platform using a scalable enzymatic synthesis strategy to produce the DNA in large quantities. Preclinical stage as of June 2023. Has raised over $536M as of June 2023. Established a strategic partnership with Moderna in March 2023.
Gensaic	Biomed	Developing M13 phage-derived particles displaying targeting molecules as a novel gene therapy vector, utilizing a high-throughput directed evolution platform to improve these phage-derived particles. Redosable since M13 phages are a part of the human virome. Tissue targets for their phage-derived particles include liver, lung, and central nervous system. As of June 2023, has raised $3.5M (grant from Cystic Fibrosis Foundation).
GenScript	Services	Services in artificial DNA synthesis, synthetic biology, antibodies, cell therapies, enzyme engineering, etc.
Ginkgo Bioworks	Services	Synthetic biology, biomanufacturing, microorganism design, enzyme engineering, etc. Acquired Gen9 in 2017.
Grove Biopharma	Biomed	Has developed proteomimetic peptide brush polymers (“bionic biologics” as they call the molecules) which act as therapeutics targeting protein-protein interactions. Their peptide brush polymers are designed to penetrate cell membranes and thus can work on intracellular targets. Their peptide brush polymers have longer half lives in vivo than traditional peptides. Has demonstrated the utility of peptide brush polymers against several different cancer and neurodegenerative disease targets in preclinical models. Raised a $30M series A as of April 2025.
HelixNano	Biomed	Developing an mRNA-based SARS-CoV-2 vaccine which might protect from all possible variants of the virus. Pivoted from original plan of developing cancer vaccines using the same technology. Co-founded by Hannu Rajaniemi, who is also a successful science fiction author. George Church is an advisor.
Humble Bee Bio	Ecotech	Identified a species of solitary bee which produces bioplastic to protect their nests and has leveraged the genetic blueprint from this bee to develop an environmentally friendly alternative to traditional plastics.
ImmuneAge Bio	Biomed	Developing ways of regenerating hematopoietic stem cells (HSCs) to treat the aging immune system and thus prevent ailments like cancer, brain aging, infections, and cardiovascular disease. Has developed a way of expanding numbers of human HSCs 1000-fold, which allows them to run high-throughput combinatorial drug screening assays for in vivo and ex vivo HSC rejuvenation. As of January 2025, working on an orally available small molecule (IA-101) which acts on mitophagy and mitochondrial biogenesis and has top indications of vaccine response, preventing respiratory infections, and mitigating post-chemotherapy immunosenescence.
Immunai	Bio-AI	Combining multi-omic single cell profiling technologies and machine learning to comprehensively map the immune system and thereby enable greatly improved immunotherapies as well as accelerate clinical trials and avoid costly failures.
Impossible Foods	Ecotech	Uses synthetic biology and biochemical engineering to develop plant-based substitutes for meat products. Their signature product is the Impossible Burger. They also make a product which mimics sausages. One notable strategy employed by Impossible Foods is production of leghemoglobin in yeast. This compound gives a meaty flavor when added to their food products. They also add other plant-based compounds to mimic the fats found in animal meat.
Imprint	Biotech	Developing experimental and computational tools to decode immunological memory in B and T cells with the aim of uncovering the causes of chronic diseases such as autoimmune conditions, long COVID, psychiatric disorders, and dementias. Hopes to pave the way for new treatments and diagnostics by uncovering the mechanisms of chronic diseases. Imprint a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Inait	Bio-AI	Aiming to develop AGI by building on work from the Blue Brain Project and Human Brain Project. Founded by Henry Markram, the pioneer behind the European Union’s (somewhat controversial) Blue Brain Project and Human Brain Project. Combines biomimetic spiking neural net (SNN) AI architectures, a brain-like learning rule discovered during Henry Markram’s studies on SNNs, and contemporary AI technologies like LLMs, CNNs, and GNNs. Goal is to use biomimetic approaches to overcome limitations found in traditional advanced AI systems, Working on architectures which possess sensory-like systems to learn from complex digital environments and to adapt and function intelligently within said environments. Has partnered with Microsoft as of March 2025. Has raised $300M according to its website.
Insilico Medicine	Bio-AI	Leveraging artificial intelligence to facilitate every step of pharmaceutical development. Has developed software to discover and prioritize novel drug targets, generate novel molecules, and design and predict clinical trials. Alex Zhavoronkov is CEO, Executive Director, and Chairman of the Board. One of the company’s lead pharmaceuticals (TNIK) represents the first AI-designed drug to reach phase II clinical trials. Has raised over $400M in funding as of June 2024.
Intellia Therapeutics	Biomed	Developing therapies which employ CRISPR gene editing technology. Has conducted some successful clinical trials using CRISPR gene therapy to treat transthyretin amyloidosis (as of February 2022, this is not yet FDA approved though). Also working on CRISPR therapeutics for engineering T cells towards targeting acute myeloid leukemia. Partnered with Regeneron, Novartis, and others. Jennifer Doudna was one of the co-founders.
Kernel	Neurotech	Neurotechnology, noninvasive brain-computer interfaces, invasive neural prostheses. Some noninvasive products anticipated to be released during 2021. Founded by Bryan Johnson who personally invested $54M. Raised an additional $53M from outside investors. Early goal is to help treat brain disease, has ambitions to enable human enhancement.
Landmark Bio	Services	Provides services for clients in cell and gene therapy development including therapeutic discovery research, process development, analytical development, quality control, GMP manufacturing, and consulting. Emerged from a public-private partnership founded by MIT, Harvard, FUJIFILM Diosynth Biotechnologies, Cytiva, and Alexandria Real Estate Equities. Their mission is to accelerate biomanufacturing of cell and gene therapies as well as to serve as a forum for biomanufacturing workforce development in Massachusetts and beyond.
Laronde	Biomed	Developing therapies which utilize circular RNAs (Laronde calls these “endless RNAs”) as expression vehicles for proteins. Such circular RNAs are much more stable and less immunogenic than linear RNAs.
Ligandal	Biomed	Peptide nanoparticles for targeted CRISPR-Cas gene therapy delivery, immunotherapy, hematological gene therapy, aging treatments. Founded by Andre Watson.
Living Carbon	Ecotech	Developing genetically modified plants (including trees) with enhanced growth, carbon capture efficiency, and bioremediation properties. Has raised over $36M and has planted over 170,000 genetically modified trees as of August 2023.
Loyal	Biomed	Developing anti-aging therapeutics for dogs including LOY-001, LOY-002, and LOY-003. LOY-001 and LOY-002 corrects for the overexpression of insulin-like growth factor 1 (IGF-1) and growth hormone (GH) found post-maturity in large dogs, they are expected to be available in 2027. LOY-002 corrects metabolic dysfunction in senior dogs, is expected to be available in 2025. As of December 2024, working on a clinical trial for LOY-002 with over 1000 senior dogs enrolled across the USA. Founder and CEO is Celine Halioua. Has raised over $125M total funding as of March 2024. Loyal’s research may pave the way for human anti-aging therapies in the future.
LyGenesis	Biomed	Allogenic cell therapy that uses host lymph nodes as bioreactors to grow ectopic replacement organs. Has developed a method for generating ectopic livers via patient lymph nodes that is in early clinical trials as of September 2022.
Mammoth Biosciences	Biomed	CRISPR-based diagnostics. Jennifer Doudna is one of the co-founders.
ManifoldBio	Bio-AI	System for barcoding protein therapeutics to enable high-throughput design and testing in complex environments, machine learning to optimize drug design. George Church is one of the co-founders.
Marblis	Ecotech	Sustainable biomaterials company with flagship product Marblis Urchinite^TM, a marble-like material made from purple sea urchins, a highly overpopulated species off the coast of California which has taken over due to rising ocean temperatures and loss of predators. Marblis Urchinite^TMcan be used as a building material for countertops, wall coverings, furniture, decor, flooring, etc. Partners with marine conservation organizations, researches ways to alleviate plastic pollution, to restore kelp ecosystems, and to leverage market-driven solutions as well as runs educational initiatives on sustainable ocean innovation. Has a biomaterials laboratory called Primitives which offers custom biomaterials R&D services and has already developed Marblis Urchinite^TM as well as seaweed-based biosensors and compostable packaging materials.
Markov Biosciences	Bio-AI	Developing deep learning tools that learn the dynamics of cellular systems by taking in a vast amount of data from genomics, transcriptomics, and more. Leveraging cutting-edge mechanistic interpretability tools to find mechanistic insights from their AI-derived simulations of cellular processes. Their mechanistic interpretability system is implemented by probing the simulations with questions in natural language, facilitating actionable insights from in silico experiments.
Medtronic	Biomed	World’s largest medical device company by revenue as of 2024 rankings, employs over 90,000 people. An American-Irish company with legal and executive headquarters in Ireland and operational headquarters in Minnesota. Operates primarily in the USA, but has some level of operation in over 150 countries. Has developed wearable and implantable pacemakers, the implantable cardioverter defibrillator, the world’s smallest pacemaker, and the world’s smallest spinal cord stimulator, an automated insulin pump, implantable drug delivery systems, and more.
Microdrop Technologies	Services	Sells instruments that can rapidly and accurately dispense liquid droplets in amounts as small as 20 picoliters using piezo-driven inkjet printing technology, also sells accurate nanoliter to microliter dispensing systems as well as instruments to automate the dispensing systems.
Micro-X	Biomed	Small, light, and fast proprietary x-ray imaging technology based on novel electronically controlled carbon nanotube emitters. Products are applied in portable medical imaging and in security. There are over 380 of their medical x-ray devices used across 35 countries. Their medical x-ray products have been used extensively in the field on Ukraine’s frontlines in the ongoing Ukraine-Russia war.
Moderna	Biomed	Biomedical technologies which utilize mRNA inside of lipid nanoparticles; application areas include drug discovery, drug development, and vaccines. Major player in COVID-19 pandemic since it was one of the first companies which developed and distributed SARS-CoV-2 vaccines to the world.
Motif Neurotech	Neurotech	Developing a small device implanted in skull bone which can perform transcranial magnetic stimulation (TMS) to treat depression and other mental health disorders, users wear a baseball cap with coils to activate the device. Unlike traditional TMS, this device does not require numerous visits to a clinic with access to bulky equipment, vastly improving accessibility. Has raised $100K as of June 2023. Jacob Robinson from Rice University is co-founder.
Nanite Bio	Bio-AI	Employing a high-throughput AI platform to predict properties of polymers and to design nanomaterials which serve as efficacious gene delivery vehicles, synthesizes and tests in vitro thousands of distinct polymer nanoparticles over a few days, uses multiplexed in vivo screening to test many polymer nanoparticles at once in animal models. Has raised $8M in funding as of June 2023.
Nautilus Biotechnology	Services	Developing a high-throughput single-molecule proteomics platform which integrates many novel techniques to decipher protein networks and thereby help accelerate basic science, new therapeutics, and new diagnostics.
Neurable	Neurotech	Developing a non-invasive brain-computer interface based on headphones that use electroencephalography to record brain signals, allowing people to control devices like phones with their minds. As of September 2022, the company appears fairly far along in its product development process and is likely to release their headphones within a year or so.
Neuralink	Neurotech	High-bandwidth brain-machine interfaces, surgical robots which implant the interfaces in a manner resembling a sewing machine. Early goal is to help treat brain disease, has ambitions to enable human enhancement. Founded by Elon Musk and others, highly publicized by Elon Musk. Has done testing on rats, pigs, monkeys, and other animals as of April 2021.
NewLimit	Biomed	Extending human longevity through epigenetic reprogramming, starting with restoring youthful function in the liver and the immune system. Has raised $40M as of May 2023. Co-founded by Coinbase CEO Brian Armstrong.
Nudge	Neurotech	Developing whole-brain focused ultrasound devices which achieve millimeter precision. Has already developed a helmet-like phased array device which can be used along with an MRI machine, allowing visualization of the effects of the ultrasound. Also has developed an MRI-based acoustic radiation force imaging technique to visualize the ultrasound focus in the brain. Has developed powerful simulation and imaging algorithm software to provide control over their device’s interactions with the brain. Running studies on patients with essential tremor, tinnitus, substance use disorders, and chronic pain (though as of September 2025, these studies are aimed at device feasibility rather than treatment). Will run studies on healthy volunteers in the future to study ways that ultrasound can influence the brain. Announced a $100M series A led by Thrive Capital and Greenoaks in July 2025.
Nvelop Therapeutics	Biomed	Developing delivery vehicles for tissue-specific targeting and gene editing; based on lentivirus-like particles with fused gene editing proteins instead of DNA inside of the envelope (as seen in publications from David Liu’s academic laboratory). Co-founded by David Liu and Keith Joung. Launched with $100M of funding as of April 2024.
Oisin Biotechnologies	Biomed	Developing senolytics which target senescent cells by triggering apoptosis only when certain genes are expressed. Has received investment from the SENS Research Foundation, the Methuselah Foundation, and the Methuselah Fund.
Olden Labs	Services	Developing technologies to automate mouse research, has released their first product: DOME cages, which use AI on 24/7 video of housed mice to track the movement of multiple mice with 99% accuracy over long time periods. DOME cages also automatically feed mice, calculate numerous behavioral metrics (e.g. total distance traveled, sleep time, average acceleration, food and water intake, number of aggressions, etc.), evaluate behavior-based health metrics, provide automated emergency alerts when problems arise, and are compatible with existing rack systems. Michael Florea is CEO and one of the co-founders.
Openwater	Neurotech	Portable medical imaging technologies which employ novel optoelectronics, lasers, and holographic systems. Wearable imaging technologies which could be 1,000x cheaper than MRI and achieve similar or better results. Has speculated that their technology might eventually allow telepathic communication. Founded by Mary Lou Jepsen.
Orchid Health	Biomed	Performs whole-genome sequencing on embryos to screen for neurodevelopmental disorders, birth defects, and chromosomal abnormalities as well as for genetic predispositions to cancers and ailments of the brain, heart, and more. Helps patients ensure that their children have a healthy future and gives them the option to not move forward with the pregnancy if the embryo may lead to an unhealthy person.
Organovo	Biomed	3D tissue bioprinting for in vivo clinical applications, in vitro tissue models for disease modeling and toxicology. Long-term goal is to print entire human organs for transplants.
Oviva Therapeutics	Biomed	Therapies for ovarian aging to aiming extend women’s healthspan and longevity. Developing a treatment to improve ovarian longevity that uses recombinant Anti-Müllerian Hormone (AMH), a first-in-class therapeutic which may delay menopause and thus exert beneficial effects on health. Daisy Robinton is a co-founder and the CEO. Raised a $11.5M seed round in May 2022.
Oxford Nanopore Technologies	Biotech	Portable nanopore sequencing devices, high-throughput desktop nanopore sequencing devices, sample preparation kits. The company states that they have the first and only nanopore DNA and RNA sequencing platform as of May 2021.
Oxgene	Biomanufacturing	Provides AAV manufacturing kits and services leveraging tetracycline enabled self-silencing adenovirus (TESSA^TM) technology to greatly enhance yields. The TESSA technique increases AAV yields by around 40-fold relative to traditional methods, increases overall infectivity of the virus particles, and is well-suited to GMP-quality production. Provides self-inactivating (SIN) lentiviral plasmids for lentivirus production with optimized safety and translation efficiency; this can increase viral yields by up to 10-fold compared to traditional methods. Provides stable lentiviral packaging and producer cell lines (based on HEK293) which facilitate consistent production of high-titer lentivirus after transfection of a viral genomic plasmid carrying a gene of interest or after stable integration of a gene of interest respectively. The lentiviral packaging and producer cell lines can be grown without animal serum in the media.
Oxitec	Ecotech	Genetically modified male insects which curb the reproduction of populations of their species in the wild, acting as a precise and environmentally friendly way of controlling dangerous pests that spread disease or destroy crops. After years of battles with activists and regulatory bodies, the company will release 750 million genetically modified mosquitos in the Florida Keys (the first time this has been done in the U.S.) with the goal of reducing rates of illnesses such as yellow fever and dengue.
Panacea Longevity	Biomed	Enhancing longevity and health using a fasting-mimetic metabolite supplementation. Early stage as of May 2021.
Panluminate	Services	Offers expansion microscopy (ExM) as a service as well as related tissue labeling (e.g. Unclearing, chromatin labels for ExM, etc.) and imaging services, can expand tissues up to 25x using their pan-ExM technology. CEO Ons M’Saad developed pan-ExM and some of Panluminate’s related technologies while working in Joerg Bewersdorf’s laboratory at Yale.
Paradromics	Neurotech	Developing surgically implanted brain-computer interface called Connexus which uses hairlike intracortical electrodes to record from 1684 channels, aims to restore communication abilities to people with severe motor impairments (e.g. amyotrophic lateral sclerosis). Interface is scalable to possibly add even more channels for future applications. Has raised a total of $88.7M as of December 2024.
Pioneer Labs	Ecotech	Not a startup company but a nonprofit research organization with a startup-like approach. Developing engineered microorganisms that may be able to grow on Mars with the future goal of terraforming, combining various types of extremophiles that individually have some of the abilities necessary for survival on Mars. Shorter term goal of green manufacturing in resource-constrained environments. CEO is Erika DeBenedictis, formerly a principal investigator at the Crick Institute. Funded by the Astera Institute as well as supported by another nonprofit founded by Erika DeBenedictis called Align to Innovate.
Precision Neuroscience	Neurotech	Developing a thin-film microelectrode array (called the “Layer 7 Cortical Interface”) which conforms to the surface of the brain and collects high-resolution data from 1024 microelectrodes. Layer 7 Cortical interface can be implanted with minimally invasive and reversible surgery, facilitates recording and stimulation, and is designed to allow paralyzed people to control computers with their thoughts. Aiming to treat conditions such as spinal cord injury, stroke, ALS, and traumatic brain injury Started clinical trials in 2023 and has (as of April 2025) tested the device in 37 patients, for which it was implanted temporarily to aid in situations like surgical removal of brain tumors. One of the co-founders is Benjamin Rapoport, who previously was part of the founding team at Neuralink. Has raised $155M as of December 2024, including series A, B, and C rounds.
Prime Medicine	Biomed	Developing CRISPR Prime editing technology as a novel therapeutic modality. David Liu and Andrew Anzalone are co-founders.
Profluent Bio	Bio-AI	AI platform for designing de novo proteins such as enzymes, gene editors, antibodies, and more. Has released the first freely available AI-generated gene editor called OpenCRISPR-1, which has similar structure to Cas proteins, yet its sequence differs by over 400 mutations compared to Cas9 and over 200 mutations compared to any known Cas protein. Has raised a total of $44M in funding as of March 2024.
Proteinea	Ecotech	Mass-produced insect larvae as an affordable way of manufacturing recombinant proteins. Early stage as of May 2021.
ReCode Therapeutics	Biomed	Has developed selective organ targeting (SORT) lipid nanoparticles, which include the four components of traditional lipid nanoparticles plus a fifth biochemically distinct lipid to facilitate bypassing of the liver and targeting of other organs such as lung and spleen. As of July 2023, has reached early-stage clinical trials for treating primary ciliary dyskinesia with inhalable SORT lipid nanoparticles that carry mRNA, is just starting early-stage clinical trials for treating cystic fibrosis with inhalable SORT lipid nanoparticles that carry mRNA, and has begun discovery-stage work on several other treatments. Has raised a total of $422M as of July 2023. Co-founded by Daniel Siegwart, a professor at the University of Texas.
Recursion Pharmaceuticals	Bio-AI	High-throughput platform for drug discovery which leverages AI and multimodal automated screening tools to achieve a cycle of homing in on useful drug molecules, narrowing the search space recursively. Has found some molecules which are now in clinical trials as of June 2023.
Rejuvenate Bio	Biomed	Developing anti-aging gene therapy using liver-directed AAVs encoding FGF21, a protein facilitates global regulation of a network of genes and helps reverse multiple conditions such as age-related obesity, diabetes, heart failure, and renal failure. Lead indication (desmoplakin arrhythmogenic cardiomyopathy) is at IND-enabling study stage as of January 2025, also at preclinical stage for other indications. Running a clinical trial for cardioprotective gene therapy to treat dogs with mitral valve disease as of January 2025. George Church is a co-founder. Has raised over $14M in funding as of January 2025.
Renewal Bio	Biomed	Developing a method that acts as an artificial womb and facilitates ex vivo production of human embryos similar to those found in a pregnancy around day 40 to 50. Have demonstrated successful proof-of-principle for making human embryos ex vivo. Aims to use the embryos as “3D bioprinters” to make tissues and organs for transplantation. The embryos may produce immune cells that could be transplanted into an older person to rejuvenate her/his immune system and facilitate longevity. The embryos may produce gonad tissues that could be transplanted into women to restore fertility and improve health. Strictly not aiming to create embryos that could develop further due to ethical issues. Likely will be able to genetically engineer the embryos to prevent formation of a head, mitigating ethical concerns. Based in Israel. Has published several high-profile scientific papers (two in Nature and one in Cell).
Repair Biotechnologies	Biomed	Developing a cholesterol degrading platform therapy which can reverse atherosclerosis. The CEO, who is known as Reason, is outspoken about the need to combat aging. Has preclinical proof-of-concept as of May 2021.
Resilience	Biomanufacturing	New manufacturing platforms to service partners for development and scaling of gene therapies, cell therapies, vaccines, protein therapies, and more. Received $800M in funding during 2020.
Retro Biosciences	Biomed	Longevity company with the goal of adding 10 years to the healthy human lifespan. Developing treatments for aging in the areas of hematopoietic stem cell reprogramming, autophagy enhancement, microglia therapeutics, tissue reprogramming, and T cell reprogramming. Sam Altman invested $180M into Retro Biosciences in 2023.
Ring Therapeutics	Biomed	Developing anellovirus as a minimally toxic and redosable alternative to existing gene therapy viral vectors. Anellovirus is a commensal human virus. Employing a platform called Anelloscope for screening of anellovirus sequences from human tissue, this then leads into to design of improved anellovirus variants.
Sanmai	Neurotech	Developing transcranial focused ultrasound devices (noninvasive) for neuromodulation to treat mental illnesses. Has published a pilot study (2023) showing that 60% of people with treatment-resistant anxiety experienced a statistically significant reduction in their feelings of anxiety. Has published a pilot study (2020) showing that 70% of people with treatment-resistant depression experienced a statistically significant improvement in certain forms of mood. As of 2025, in the process of performing a clinical trial with Acacia Mental Health clinic to determine if multiple sessions extend anxiety reduction effect. Raised a $12M series A in June 2025 from Reid Hoffman (LinkedIn co-founder and wealthy individual).
Sarepta Therapeutics	Biomed	A large medical biotechnology company with 4 FDA-approved therapies and 40 investigational therapies under-development (as of September 2025). Their FDA-approved therapies include three antisense oligonucleotides (made with phosphorodiamidate morpholino oligomer backbones) as well as the AAV gene therapy Elevidys, all medicines for treating Duchenne muscular dystrophy. The investigational therapies are aimed at treating Duchenne muscular dystrophy, limb-girdle muscular dystrophies, Charcot-Marie-Tooth disease, and some CNS-related disorders. The cost of Elevidys is $3.2M per patient (one-time treatment). Elevidys has endured significant public controversy due to several patient deaths (acute liver failure) in 2025, which led to a hold on the therapy as requested by the FDA. The hold on Elevidys was lifted by the FDA in July 2025, but concerns remain. Additionally, the European Medicines Agency has recommended against marketing Elevidys in the European Union.
Science	Neurotech	Developing a device-therapy combination to restore sight in people who have lost photoreceptors but retain retinal ganglion cells. Leveraging optogenetic gene therapy to give retinal ganglion cells the ability to respond to light as well as an implantable device that fits over the retina and stimulates the modified retinal ganglion cells with appropriate wavelengths to reproduce vision. Has an in-house foundry which can provide custom electronics fabrication as a service to interested parties. Also developing a new type of brain-computer interface which uses an external device containing living neurons that interface with the brain tissue, are activated via optogenetic stimulation in the external device, and are recorded by electrodes in the external device.
Sherlock Biosciences	Biomed	CRISPR-based diagnostics. Feng Zhang is one of the co-founders.
Siren Biotechnology	Biomed	Developing AAVs encoding cytokines to induce the immune system to attack solid tumors. Planning a first clinical program which will use cytokine-encoding AAVs to treat gliomas via local delivery, taking advantage of the brain’s immune-privileged status to avoid anti-AAV immunity. A low dose of AAVs is injected directly into tumors, expresses cytokines which kill the cancer cells as well as attract the innate immune system (e.g. macrophages, natural killer cells) to further eradicate the cancer. Nicole Paulk (formerly a UCSF professor) is the CEO, founder, and president. Has raised $25.6M as of January 2025.
Somalogic	Biotech	Proteomics platform called SomaScan for protein biomarker discovery which aids researchers in the development of new diagnostics. SomaScan is an aptamer-based platform which can simultaneously measure 7,000 protein biomarkers. Founded by Larry Gold, who is the inventor of SELEX.
SpiNNcloud	Bio-AI	Developing neuromorphic supercomputers so that AI can take advantage of biologically-inspired hardware architectures. Has built SpiNNaker and SpiNNaker2 high-performance computing clusters using their neuromorphic chips; these systems are highly scalable and energy efficient. The original SpiNNaker system was developed as part of the Human Brain Project; both systems are well-suited to running biologically realistic neuroscience simulations in real time.
SpyBiotech	Biomed	Developing a vaccine against human cytomegalovirus using virus-like particles equipped with their SpyTag-SpyCatcher molecular glue technology. As of June 2024, is in the process of a phase I clinical trial for their vaccine against human cytomegalovirus. Has licensed the SpyTag-SpyCatcher technology to a variety of research groups working on vaccines for cancer, chronic diseases, viral diseases, bacterial diseases, parasite diseases, and veterinary diseases. Mark Howarth, who originally developed the SpyTag-SpyCatcher technology in his academic lab, is a co-founder.
Strateos	Services	Offers R&D services through remotely controlled automated laboratories. Has extensive automated equipment for research in drug discovery, synthetic biology, imaging, cell and gene therapy, etc.
Synchron	Neurotech	Endovascular brain-computer interfaces as a minimally invasive approach for neural prosthetics, neuromodulation, and neurodiagnostics. Has developed the strentrode, an endovascular electrode array that can record or stimulate neurons from within blood vessels. As of September 2022, a technology called brain.io (that employs stentrodes) is in early clinical trials and gives paralyzed patients the ability to control digital devices.
Synthego	Services	CRISPR genome engineering services, custom cell lines, custom screening libraries, CRISPR reagents and kits, aiding both academic researchers and clinical drug developers.
Systemic Bio	Biotech	Develops vascularized organ models in hydrogels as tools for accelerating and improving preclinical drug testing.
Syzygy Plasmonics	Ecotech	Developing a photocatalytic reactor system which leverages a nanoparticle-based plasmonic photocatalyst. The photocatalyst consists of a larger light-harvesting plasmonic nanoparticle decorated with smaller catalytic nanoparticles. Their first product will be a clean hydrogen fuel production system which does not rely on petroleum. More of a chemical engineering company than a biotechnology company, but their technology may eventually have applications in biology.
Tahoe Therapeutics	Bio-AI	Developing AI virtual cell models to help find drugs for treating cancer. Working with data from perturbative interactions between single cells and drugs as well as data from drug-patient interactions. Has built an open-source foundation model (Tahoe-100M) which was trained using 100M single cell data points and 60,000 drug-patient interactions. Working on larger models to continue towards the goal of finding clinical leads to translate. Has raised a $30M series A (as of August 2025) to build a model mapping between 1B single-cell datapoints and 1M drug-patient interactions.
Tektonyx Bio	Biotech	Developing new protein therapeutics using genetically recoded bacteria with expanded genetic alphabets which include noncanonical amino acids. Has several journal publications from 2015 to 2021.
Tessera Therapeutics	Biomed	Developing gene writing technology for therapeutics. Characterizing a database of over 100,000 candidate mobile genetic elements to use in their technologies; using these to develop a toolkit for single nucleotide edits, correcting pathogenic alleles, replacing whole exons, and introducing whole genes. Leveraging target primed reverse transcription (TPRT) to engineer the genome, this is done with an RNA template and a gene writer protein, these components come from retrotransposon systems. TPRT gene writers bind and nick DNA before using reverse transcription to write into the genome (no double strand breaks). Also developing DNA gene writers for stably integrating large pieces of DNA into the genome; these can be delivered with lower doses of AAVs compared to traditional gene therapies. Has developed proprietary lipid nanoparticles for delivery of template RNAs along with RNAs encoding the gene writers to the liver, hematopoietic stem cells, and T cells. Seeking to address monogenic diseases, provide genetic treatments for prevalent diseases, and develop both in vivo and ex vivo cancer treatments. Has raised over $500M in total funding as of April 2022, more recently (December 2024) completed an agreement with the Bill and Melinda Gates Foundation to receive an additional amount of up to $50M.
Terrain Biosciences	Services	Leverages next-generation AI to design libraries of optimal RNA sequences (improved manufacturability, stability, expression quality, immunogenicity, durability, and targetability) for customers, has a rapid manufacturing pipeline to produce the RNAs. Aids customers in designing lead RNAs at early stages of development, helps via high-quality manufacturing for later (clinical) development. Co-founded by Patrick Hsu, Jonathan Gootenberg, and Omar Abudayyeh. Raised $9M in seed funding as of February 2025.
The Far Out Initiative	Biotech	Public benefit corporation developing technologies to mitigate biological suffering as inspired by philosopher David Pearce’s Hedonistic Imperative manifesto. Investigating cases of pain insensitivity where people experience very little or no pain while still having an instinctive capacity to avoid actions which may cause bodily harm; aiming to use the genetics of such people to develop a gene therapy that confers similar benefits. Working on germline engineering of farm animals and feed animals to minimize their capacity for suffering. Carefully evaluates the ethics of its proposed technologies and similar emerging technologies in order to hopefully move the world towards less suffering overall without accidentally exacerbating mistreatment of animals, etc. Early stage as of January 2025.
Tidal	Ecotech	Provides autonomous underwater robotic camera system equipped with AI computer vision technology along with environmental sensors which help fish farmers keep track of the growth, behaviors, and health of their fish and to monitor the water’s salinity, temperature, etc. The systems can furthermore facilitate real-time biomass monitoring, sea lice detection, autonomous feeding, and thus inform the decision making of fish farmers. Much of the data collected by the computer vision system occurs on timescales of milliseconds, faster than the human eye can track. Has expanded use of their technology beyond Norway to customers across the globe. Also working to leverage its systems towards ways of protecting the Earth’s oceans. Started out as a project at Alphabet’s X Moonshot Factory.
Tilibit Nanosystems	Services	Service which gives researchers predesigned and custom DNA origami nanostructures, including ones with chemical modifications. Founded by Hendrik Dietz, who was CEO from 2012-2014. He is now a scientific advisor.
Topas Therapeutics	Biomed	Developing Topas Particle Conjugates (TPCs), which consist of nanoparticles linked to immunogenic epitopes involved in selected autoimmune diseases; TPCs target liver sinusoidal endothelial cells (LSECs) and induce antigen-specific immune tolerance, thus treating specific autoimmune conditions. Targeting LSECs with TPCs results in antigen-specific tolerance via induction of regulatory T cells. As of January 2025, has shown positive results from a phase 2a trial for treating celiac disease. Has also advanced a treatment for the rare disease pemphigus vulgaris to phase 2 clinical trials. Has raised a total of $70M as of January 2025.
TreeCo	Ecotech	Developing CRISPR gene editing technology to create enhanced trees with improved characteristics for applications in timber, pulp and paper, and biofuels as well as for sustainability. For the sustainability applications, they are working on improved frost tolerance, drought tolerance, and disease resistance.
Turbine AI	Bio-AI	Predictive computational models of cancer cells, the “Simulated Cell^TM” platform, performing in silico experiments to test millions of drugs. Has partnered with Bayer, AstraZeneca, and others for drug development efforts.
Twist Bioscience	Biomanufacturing	Artificial DNA synthesis services. Synthetic biology towards insulin manufacturing in yeast, scalable spider silk manufacturing, combating malaria, and DNA data storage. Emily Leproust is a co-founder.
Vault Pharma	Biomed	Protein vault nanocompartments as a drug delivery platform to treat cancers and other diseases, protein vaults as a vaccine platform. Co-founded by Leonard Rome.
VectorBuilder	Services	Services in vector cloning, virus packaging, library construction, cell lines, etc.
Verve Therapeutics	Biomed	Developing CRISPR base editing therapies to turn off key genes (e.g. PCSK9 and ANGPTL3) involved in atherosclerotic plaque formation and thus to combat cardiovascular disease. The delivery mechanism involves lipid nanoparticles carrying gRNA and mRNA encoding a base editor protein. Has potential to save tens of millions of lives due to the status of heart disease as one of the most common causes of death. Early clinical trials began in July 2022.
Virica Biotech	Biomanufacturing	Helps in biomanufacturing of viral vectors through utilizing Viral Sensitizers, a library of small molecules which inhibit cellular antiviral defenses and thus increase yields of viruses from producer cells by around 5-10x. Provides custom services in aiding client biomanufacturing process development by incorporating Viral Sensitizers. Has raised $1.1M as of July 2023.
Xaira Therapeutics	Bio-AI	Leveraging machine learning data generation to develop a drug discovery platform and therapeutic products. Has received $1B of funding as of April 2024, though was recently announced and is still ramping up. David Baker is a co-founder. Led by Marc Tessier-Lavigne, former CSO of Genentech. Staff includes the scientists who developed RFdiffusion and RFantibody in David Baker’s lab.
YourChoice Therapeutics	Biomed	Developing a daily non-hormonal male birth control pill which is thus far (i) 99% effective and 100% reversible in mice and (ii) after 2 weeks decreases sperm count in primates to a level below fertility threshold as defined by NIH Contraceptive Development Program chief. Has completed a phase 1a clinical trial in human patients to evaluate safety and pharmacokinetics as reported by a 2025 publication. Their drug blocks spermatogenesis by inhibiting sperm progenitor cell division and by inhibiting release of mature sperm from the seminiferous tubules. Raised a $15M series A as of July 2022.
Zetta.ai	Neurotech	Advancing connectomics via computational reconstructions of neuronal wiring diagrams from image data. Offers a wide variety of automatic image reconstruction services to neuroscience laboratories. Created the automated AI reconstruction software behind reconstruction of the cubic millimeter of mouse cortex from MICrONS as well as reconstruction of the adult Drosophila brain from FlyWire. Working closely with BRAIN CONNECTs, a ten-year effort with the US BRAIN Initiative aiming to map a whole mouse brain.
Zymergen	Ecotech	Synthetic biology, metabolic engineering, biomanufacturing of materials and compounds as a substitute for chemical engineering practices.
4D Molecular Therapeutics	Biomed	Using high-throughput screening and recombination methods to develop novel AAV serotypes that evade immune responses and that target and transduce specific organs. Clinical trials for several new AAV vectors that treat pulmonary, cardiac, and eye diseases are ongoing as of September 2022
10x Genomics	Services	Spatial transcriptomics, genomics, proteomics, immune cell profiling, etc. Acquired ReadCoor and Cartana in 2020.
64x Bio	Biotech	High-throughput screening and computational design of new mammalian cell lines for manufacturing gene and cell therapies. George Church and Pamela Silver are among the co-founders.