A Type II-B Cas9 nuclease with no detectable off-targets and reduced chromosomal translocations in vivo.

Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as components of CRISPR-Cas9 genome editing and engineering platforms. However, they exhibit promiscuous nuclease activity that induces undesired mutations and chromosomal rearrangements. With SpCas9-based therapeutic gene editing entering clinical trials, such unintended editing outcomes are of increasing concern. Although several strategies for mapping CRISPR-Cas9 off-target effects have emerged, they have limitations that impact their sensitivity. To address this problem, we developed an off-target assessment workflow that uses duplex sequencing, and showed that this strategy increases the sensitivity for CRISPR-Cas9 mutation detection by one order of magnitude. This enabled us to identify previously unreported off-target mutations associated with wild-type SpCas9 treatment in an in vivo humanized PCSK9 mouse model of hypercholesterolemia. In an effort to reduce off-target risks of CRISPR-Cas9, we performed a bioinformatic search and identified a Cas9 variant of the II-B subfamily with intrinsic high fidelity, Cas9 from Parasutterella secunda (PsCas9). PsCas9 showed improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. Thus, PsCas9 offers an alternative to SpCas9 for research and clinical use. In the future we believe that the use of the duplex sequencing workflow will enable a more sensitive assessment of CRISPR-Cas9 editing outcomes and that PsCas9 will provide a more precise genome editing tool for research and for clinical applications.