Spotlight: New Hypercompact Megaphage Cas Enzyme (CasΦ) Shows Efficient Gene Editing

By Ishani Dasgupta, Ph.D.

Gene editing has emerged as a very promising tool that enables scientists to add, delete, or modify genes at targeted genomic locations. These alternations are triggered by engineered nucleases that induce a double-stranded break (DSB) in the desired genomic locus, leading to activation of efficient DNA repair mechanisms present in all organisms. This technology has proven instrumental in the treatment of a diverse range of genetic diseases.

CRISPR/Cas Systems

Among the different engineered nucleases, the CRISPR/Cas system revolutionized genome engineering most effectively. This machinery was first discovered as a component of the bacterial adaptive immune system. Since then, the type II CRISPR/Cas system has emerged as the most widely used and robust nuclease for genome editing studies.

This RNA guided type II complex consists of two components, a Cas9 endonuclease and a guide RNA (gRNA). The gRNA constitutes a ~20-nucleotide spacer sequence, called CRISPR RNA (crRNA), which is complementary to the target DNA, thus defining the genomic target to be edited and a scaffold sequence required for Cas binding namely, tracrRNA. The gRNA sequence confers specificity to the CRISPR/Cas system for targeted gene editing. Additionally, a protospacer adjacent motif (PAM) sequence immediately downstream of the target site also determines the system’s specificity and serves as a binding signal for the Cas protein.

Cas nucleases isolated from different bacterial species recognize respective PAM sequences. The commonly used and well-characterized Cas9 endonuclease is from Streptococcus pyogenes, which requires a 5’-NGG-3’ PAM sequence immediately downstream of the target site for binding. Cas9 and gRNA forms a ribonucleoprotein complex (RNP), facilitated by gRNA scaffold (tracrRNA), while the spacer region (crRNA) is free to interact with the target DNA. Once the RNP complex binds to the putative target DNA, the gRNA anneals to the target, and Cas9 undergoes a conformational change and cleaves the target strand at ~3-4 nucleotides upstream of the PAM sequence and the non-target strand resulting in a DSB at the desired genomic locus. The Cas9-mediated DSB can then be repaired by either nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathway.

Despite the breakthroughs of CRISPR/Cas9 based genome engineering, some unmet challenges intervene with its therapeutic gene editing potential. Recombinant adeno-associated vectors (rAAVs) serves as an excellent vehicle for CRISPR-Cas9 delivery of rAAVs for gene therapy in various diseases like diseases α-1 antitrypsin deficiency, cystic fibrosis, muscular dystrophy, Pompe disease, Parkinson’s disease, hemophilia B, etc. Although rAAVs serve as a robust gene delivery vector, its limited packaging ability restricts the transgene’s size. As a result, with compact CRISPR-Cas systems available, there would be enough space for DNA repair donor template, proteins fused to Cas9, and additional elements that regulate gene-editing potential. This has prompted researchers to be on the constant lookout for smaller Cas proteins.

CasΦ Makes Entry

While there are smaller orthologues of Cas9 available, a recent study by Basem Al-Shayeb doctoral student working in Jillian F. Banfield’s laboratory at the University of California, Berkeley, unraveled a CasΦ-containing megaphage that belonged to a group called Biggiephage. So far, all the established CRISPR-Cas systems were discovered in bacteria and Archaea as a component of their immune system to protect against viruses, but this was the first-ever and only study that found a novel CRISPR-Cas system in viral genomes. CasΦ is about half the size of Cas9, around ~70-80kDa, making it a lot easier to deliver into cells as compared to Cas9. Metagenomic sequencing studies revealed the gene encoding CasΦ and shares remote homology with the Type V Cas system.

Further studies led by Jennifer A. Doudna’s group in UC Berkeley were conducted to characterize its functionality. The ability of CasΦ to target foreign plasmid DNA in Escherichia coli enabled its protective function. Akin to Cas9, the more compact CasΦ cleaves double-stranded DNA with similar efficiency and selectivity. The minimal PAM requirement for this protein renders it more accessible for targeting a wider array of sequences ranging from human embryonic kidney cells to Arabidopsis thaliana plant cells.

Another property of CasΦ that has a clear advantage over the existing compact Cas proteins is its streamlined ability to combine multiple functions into a single protein, making it an attractive tool for genome engineering and enabling easier vector-mediated delivery. The other Cas enzymes have separate sites for snipping DNA and crRNA processing. But this study reported that CasΦ uses a single site for both the functions. IGI executive director Jennifer Doudna mentioned, “When we think about how CRISPR will be applied in the future, that is one of the most important bottlenecks to the field right now: delivery. We think this very tiny virus-encoded CRISPR-Cas system may be one way to break through that barrier.”

Overall, this new study highlights the emergence of the hypercompact CasΦ from the megaphages as a valuable addition to the CRISPR toolbox, rendering them as a potential frontline robust tool for genome engineering.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: CRISPR C-to-G Base Editors: A Therapeutic Promise

References

1. https://science.sciencemag.org/content/369/6501/333
2. https://news.berkeley.edu/2020/07/16/megaphages-harbor-mini-cas-proteins-ideal-for-gene-editing/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939442/
4. https://www.tandfonline.com/doi/full/10.1080/10717544.2018.1474964

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