Abstract: CRISPR-based gene editing shows great potential for the curative treatment of genetic diseases. Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as components of CRISPR/Cas9 genome editing platforms. However, they may lead to unintended mutations and chromosomal rearrangements that pose a major safety risk for clinical applications. Current strategies for mapping CRISPR/Cas9 off-target effects 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 undetected off-target mutations associated with 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, which we named Specific Off-Target/ON-target (SpOT-ON) discriminator. SpOT-ON Cas9 showed improved specificity as compared to SpCas9 across multiple tested sites both in vitro and in vivo, including the PCSK9 site. Thus, SpOT-ON Cas9 offers an alternative to SpCas9 for research and clinical use. Going forward, we expect that combined use of the duplex sequencing workflow we developed and SpOT-ON Cas9 will enable more sensitive assessment of CRISPR/Cas9 editing outcomes, as well as more precise genome editing tools and treatments.
