We’ve already seen how CRISPR-Cas9 is making the headlines with its utility in human embryonic stem cell editing, but now it’s getting a makeover to make it even more attractive for use in humans.
Current CRISPR-Cas9 genome editing technology is faced by two translational limitations. The first is targeting, where PAM sequences and sgRNA design technicalities limit the targets Cas9 can cleave, while also spurring the quest for Cas9 orthologs that employ alternate PAM sequences. Another challenge faced is delivery, since current systems employ adeno-associated virus (AAV) vectors to ferry foreign genetic material into human cells.
AAV vectors are befitting for this purpose, because of their low immunogenicity, reduced oncogenic risk, and versatile serotype specificity. However, AAV vectors can only carry cargos of around 4.5 kb in length. This cargo size limitation makes AAV vectors unsuitable for packaging the famed precise genome editing enzyme Cas9 from Staphylococcus pyogenes (SpCas9, size ~4.2 kb) and its guide RNA (sgRNA) and effectively delivering the desired cargo into human cells.
To overcome this size limitation and put CRISPR-Cas9 a step closer to therapeutic applicability, a team from MIT and Harvard led by Feng Zhang undertook a mission to search for a smaller Cas9 enzyme that can be effectively packaged in AAV and conveyed into desired target human cells without problems.
First, the determined team explored the structure of over 6,000 Cas9 orthologues to determine the key discrepancies between the longer and shorter Cas9 enzymes. The team found that shorter Cas9 enzymes have abridged CheY-like phosphoacceptor (receiver or REC) domains. From the pool of short Cas9 enzymes with a number of different PAM sites, the talented team chose six orthologues from Staphylococcus aureus (SaCas9, size ~3 kb) for further exploration and found that:
- SaCas9 achieves highest editing efficiency with gRNAs between 21 and 23 nt long, which is a little higher than SpCas9 gRNA requirement.
- Although being 1-kb shorter, SaCas9 edits the genome with similar efficiencies to that of SpCas9
- AAV packaged SaCas9 and its guide RNA modified cholesterol regulatory gene Pcsk9 in mouse liver greater than 40% within one week of injection.
- The modification resulted in significant reductions in serum Pcsk9 and overall cholesterol levels.
Assessing the genome-wide targeting specificity of the SaCas9 system, the team concludes that “SaCas9-mediated in vivo genome editing has the potential to be efficient and specific”.
For further exploration how this alternative Cas9 is helping CRISPR on it’s journey to the clinic venture over to Nature, April 2015.