A Compact Cas9 Emerges From the Human Microbiome

CoCas9, a 1004 amino acid protein derived from an uncultivated Collinsella species, stands out due to its small size and high editing efficiency, making it particularly suitable for AAV delivery systems. The study demonstrated that CoCas9 could be efficiently packaged with its gRNA into an all-in-one AAV vector, achieving effective in vivo editing in mouse models.

This feature is significant as the smaller size overcomes the typical limitations of delivering CRISPR components, which often restrict the use of larger Cas proteins like SpCas9 in clinical applications.

The research team conducted extensive analyses to characterize CoCas9, including its protospacer adjacent motif (PAM) preferences and off-target effects. These investigations revealed that CoCas9 has a unique and slightly relaxed PAM requirement compared to more commonly used Cas9 variants, which may increase its utility for a broader range of genomic targets.

Off-target (OT) analyses at four loci (HPRT, VEGFAsite2, ZSCAN2 and Chr6) demonstrated that CoCas9 produced considerably fewer OT cleavages than SpCas9. For example, at the OT benchmark locus VEGFAsite2, CoCas9 produced 19-fold fewer off-targets than SpCas9, while cleavages at the specific site by CoCas9 were 180-fold higher.

CoCas9 was identified by exploring a vast database containing over 154,000 microbial genomes reconstructed from more than 9,400 human-associated metagenomes. This process involved interrogating metagenome-assembled genomes to uncover a diverse array of 17,173 type II CRISPR-Cas loci, from which the compact and highly active CoCas9 nuclease was singled out due to its desirable properties for genome editing applications.

The study was led by Anna Cereseto and Nicola Segata at the University of Trento, Italy, and it was published in Nature Communications last week. You can read it here.