New Cas9 Alternatives Give Mammoth Biosciences an Edge Over Other CRISPR companies

By Eduardo Longoria

Genome editing is the process by which researchers make targeted changes to an organism’s DNA. While there are various technologies for genome editing, the process has become dramatically easier since the advent of CRISPR/Cas9 technology in 2012. The Cas (cellular apoptosis susceptibility) proteins are part of the CAS/CSE protein family, including yeast chromosome-segregation protein, CSE1. They are relatively easy to target specific DNA sequences and work in many organisms.

Cas9 Alternatives

While only a few Cas proteins see regular use in genomic medicine, the CAS/CSE family is vast and has a few members that could be superstars in treating hereditary diseases. Here are a few notable members:

Cas12a

Able to cut double-stranded DNA at places that Cas9 can’t, and since it leaves ragged edges, Cas12a is perhaps easier to use when inserting a new gene at the DNA cut.

Cas13

Previously known as C2c2, the effector protein targets and cleaves invading nucleic acids from viruses in type VI CRISPR-Cas systems. The CRISPR-Cas13 cleaves RNA rather than DNA and was discovered in L. shahii, a Leptotrichia bacteria species. Members of Cas13, Cas13a, and Cas13b are under development for therapeutic gene correction at the RNA level and detection of viral pathogens.

dCAS9

In addition to the naturally occurring Cas proteins, there are also mutant versions made to get around some of the limitations that natural proteins have. Dead endonuclease Cas9 (dCas9) can increase gene expression by using transcription factors, activators, repressors, and histone modifiers.

SpCas9: Limitations and Origin

Despite the great potential and current success of some CAS/CSE family proteins, SpCas9 stands head and shoulders above the crowd by sheer notoriety. However, it is not without its shortcomings. SpCas9 comes from a pathogenic bacterium called Streptococcus pyogenes. Some reports suggest that using SpCas9 for genome editing in humans may lead to dangerous immune reactions (Ferdosi et al. 2019). However, others have questioned the importance of this finding.

Moreover, SpCas9 is 1368 amino acids (aa) long and can be difficult to fit into standard delivery vehicles and hard to get into target cells and tissues. Finally, it requires the presence of a specific DNA sequence known as a “PAM” (protospacer adjacent motif) to target an adjacent sequence for genome editing. The need for this sequence (5’-NGG-3’) restricts the number of sites it can edit.

Cas14 and CasΦ Discovery

Given the restrictions of SpCas9, researchers have been using metagenomics to discover new Cas proteins for genome editing and other applications. Briefly, this process makes use of DNA sequence databases sampled from many environments. Researchers sift through these sequences to find signatures of CRISPR systems. Once found, they isolate these systems and determine if they are functional. Some functional systems can later guide genome editing.

Using metagenomics techniques, Mammoth Biosciences Co-Founder and CSO Lucas Harrington and his colleagues found and characterized Cas14. One of the Cas14 protein family members came from an archaeon sampled by the Banfield Lab from groundwater near Rifle Colorado (a historical uranium and vanadium mine). By studying these microorganisms, researchers hope to understand how residual contamination from the mines affects the environment. Cas14 is theorized to be used by the archaeon to defend against single-stranded DNA viruses.

Similarly, Co-Founder and Nobel Laureate, Dr. Jennifer Doudna, and colleagues recently discovered and characterized CasΦ. This Cas protein family comes from “phages,” the viruses that infect bacteria. In particular, CasΦ came from a group of phages with large genomes known as “Biggiephages.” These phages have been found in metagenomic samples from a variety of locations. It’s believed that phages use CRISPR systems to disrupt certain bacterial processes. They may also prevent other phages from infecting the same bacterial host.

Cas14 and CasΦ Advantages

Cas14 and CasΦ both have beneficial properties for genome editing that may make them more useful than SpCas9. Neither Cas14 nor CasΦ comes from a human pathogen and is thus less likely than SpCas9 to trigger immune responses in humans.

Both Cas14 and CasΦ are quite small, with members of the Cas14 family at around 500 aa and members of the CasΦ family are slightly larger at around 750 aa. This means proteins from both families should be easier to deliver to target tissues than SpCas9.

Finally, both Cas14 and CasΦ have less restrictive PAM requirements than SpCas9. They use T-rich sequences as PAMs with flexible amino acid requirements. For example, one of CasΦ’s PAMs is 5’-TBN-3’ (where B is G, T, or C). These loose PAM requirements open more DNA sequences to genome editing by Cas14 and CasΦ, making them more useful than SpCas9. We hope these proteins will make it easier to achieve a variety of biotechnological goals.

Competition in the Market

Apart from the few notable companies such as Ionis, Intellia, Editas, and CRISPR Biosciences, there are roughly 10 smaller companies involved in deep research into CRISPR technology. Mammoth Biosciences can take advantage of the Cas14 and CasΦ systems to work on therapies for diseases that Cas9 cannot. For example, Mammoth could harness Cas14 to research single-stranded DNA viruses and their diagnosis as well as SNP genotyping.

Suppose Mammoth manages to make this strategy work, they can avoid having to compete against the likes of Intellia and their massive financial resources to work on gene editing with Cas9. Mammoth Biosciences has only received $68.2 million worth of funding since its creation in 2017. The company is privately held and has a post-money valuation in the range of $100M to $500M as of Jan 30, 2020. The ability to bring both Cas14 and CasΦ to bear against genetic conditions that would otherwise be outside of the reach of genetic treatment gives this smaller and privately held company an edge over the rest. Using their new IP, the reputation of their founders and the recent Nobel prize awarded to Dr. Jennifer Doudna, Mammoth has a competitive leg up in the market and, should the need arise, a strong position from which to fundraise.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: Mammoth Biosciences Signs Deal with UC Berkeley for Novel Micro CRISPR Protein

References

1. https://mammoth.bio/2019/12/04/how-do-researchers-expand-the-crispr-toolkit/
2. https://pubmed.ncbi.nlm.nih.gov/32051592/
3. https://pubmed.ncbi.nlm.nih.gov/30337455/
4. https://pubmed.ncbi.nlm.nih.gov/32675376/

Author

Eduardo Longoria