Expanding CRISPR repertoire using CjCas9 as a smaller editing tool

Abstract

The field of gene editing continues to expand significantly and is entering a time of unprecedented utility. Academia and industry look to conquer genetic-based disease with viral and non-viral-delivered CRISPR-Cas9. The most widely used Cas9 protein is derived from Streptococcus pyrogenes (SpCas9), which lends itself to being too large for AAV viral delivery. Cas9 orthologue proteins have diverse size and dependent on bacteria of origin. This diversity has given rise to Cas9 proteins smaller in size while maintaining gene editing abilities. In this article, authors have focused on the use of CjCas9, whose smaller size allows for packaging in AAV and maintains high on-target gene editing. The locus APOC3 was identified for eventual targeting/integration in humans where cardioprotective properties are predicted. To confirm in vivo targeting of this locus, a humanized mouse model was developed due to the absence of the APOC3 locus in mice. These studies looked to answer long-standing questions on integrated gene stability, promoter/low gene integration, and the duration of therapeutic efficacy of the integrated gene.