Evolved Cas9 Facilitates CRISPR Access to Previously Unexplored Genomic Sites

By Ruchi Jhonsa, Ph.D.

Edited by Rajaneesh K. Gopinath, Ph.D.

Today, CRISPR genome-editing has become a household name and has progressed rapidly since its commercialization in 2012. Several improved versions are developed by the minute and the technology is poised to dominate therapeutic interventions in the future. It is rather interesting to note that, the fundamental principle behind CRISPR based gene editing was discovered when scientists stumbled upon a bacterial defense mechanism.

Several bacteria and archaea have evolved to protect themselves from viral attacks through intricate machinery that chops viral DNA into pieces. However, CRISPR machinery is not random. DNA sequences from previous infections are stored in the CRISPR locus and get transcribed into CrRNAs that bind to incoming viral DNA in the event of a subsequent invasion. The viral DNA is identified through 2-6 base pair long DNA sequences called protospacer adjacent motif (PAM) embedded throughout the viral genome.

Scientists soon realized they have hit gold with this discovery. Until that time, they were developing cumbersome technologies such as Zinc Finger Nucleases (ZFNs) and Transcription activator-like effector nuclease (TALENs) for genome editing. CRISPR was a relatively simpler technique that allowed them to tweak the genomes whichever way they want it. By designing a guide RNA, scientists could target a CRISPR associated endonuclease (Cas9) to edit a desired region in the genome. When the technology was commercialized, people expected CRISPR to cure thousands of genetic diseases at a rapid pace. Although not impossible, to achieve this goal, the gene editor has to overcome several limitations, the requirement of the PAM sequence being one.

Now investigators at the Massachusetts General Hospital (MGH) have modified the Cas9 derived from Streptococcus pyogenes (SpCas9) to increase the access of the editing machinery to previously inaccessible genomic sites. The two variants, SpG and SpRY engineered at MGH can edit DNA at efficiencies not achievable with conventional CRISPR-Cas9 enzymes. Moreover, SpRY has an additional feature that makes it limitless and unbiased towards several PAM sites.

Limitations of CRISPR

CRISPR is a two-component gene-editing tool that uses guide RNA to recognize the target sequence and Cas9 to cut the DNA. However, it does not cut all the DNA sequences because of the PAM requirement constraint. Not all target genomic locations have a PAM sequence adjacent to them and this barrier prevents the accurate positioning of CRISPR target sites.

SpCas9 is the most commonly used Cas9 that recognizes just a 3 base pair PAM sequences NGG, where N stands for any nucleotide. For years, scientists have worked around making Cas9 less NGG biased by either substituting different amino acids at the PAM-Cas9 interaction site or by directed evolution. While these developed Cas9 variants were able to recognize other PAM sequences, it did not completely relax Cas9 from the PAM requirement.

The New Development

For a CRISPR system to target a large number of genomic sites, it is important to remove the dependency of SpCas9 on a requisite PAM. In this new study published in the journal, Science, Dr. Benjamin P. Kleinstiver and team from MGH’s Center for Genomic Medicine attempted to do that. They show that amino acid substitution in the sites necessary for PAM-Cas9 interaction relaxes or almost entirely removes the requirement of NGG PAM for SpCas9. This extends targeting to sites with NGN and NAN PAMs and many sites with NCN or NTN PAMs.

So far, the evolved Cas9 variant’s unbiased targeting and high efficiency have been shown in lab-grown kidney cells but its utility is still unproven across different applications and delivery contexts. Nevertheless, the system looks promising and is suggested for targeting NGH (where H is A, C, or T) and NAN PAMs than NCN and NTN PAMs. Dr. Kleinstiver said, “By nearly completely relaxing the requirement for the enzymes to recognize a PAM, many genome editing applications are now possible. And since almost the entire genome is targetable, one of the most exciting implications is that that the entire genome is ‘druggable’ from a DNA-editing perspective.”

CRISPR in the News

CRISPR’s rise as a potential medical tool happened in a remarkably short time. CRISPR based gene editing has been used to fight diseases such as Duchenne Muscular Dystrophy and repair mutations in multiple cancer types previously. Last year, CRISPR therapeutics joined hands with Vertex Pharmaceuticals in treating blood disorders like beta-thalassemia and sickle cell disease using this technology. The most exciting news came early this month when a landmark CRISPR trial conducted by Editas Medicine marked the first-ever gene-editing inside the human retina correcting gene mutation for congenital blindness. Though the outcome of this trial is still pending, we hope that it has a happy ending.

Related Article: Evolved Base Editor Surpasses Predecessors with High Efficiency and Versatility

References

1. https://www.ncbi.nlm.nih.gov/pubmed/32217751
2. https://www.ncbi.nlm.nih.gov/pubmed/26098369
3. https://www.ncbi.nlm.nih.gov/pubmed/26422227
4. https://news.harvard.edu/gazette/story/2020/03/removing-the-constraining-requirements-at-gene-editing-site/

Author

Ruchi Jhonsa