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.

酿脓链球菌Cas9（Streptococcus pyogenes Cas9，SpCas9）及其衍生酶作为CRISPR-Cas9基因组编辑与工程化平台的核心组分被广泛应用。然而其存在非特异性核酸酶活性，可诱导不必要的突变与染色体重排。随着基于SpCas9的治疗性基因编辑进入临床试验阶段，这类非预期编辑结果愈发受到关注。尽管目前已涌现出多种用于解析CRISPR-Cas9脱靶效应的策略，但这些方法均存在影响检测灵敏度的局限性。为解决这一问题，本研究开发了一种基于双工测序（duplex sequencing）的脱靶评估流程，实验结果表明该策略可将CRISPR-Cas9突变检测的灵敏度提升一个数量级。这使得本研究得以在高胆固醇血症的体内人源化PCSK9小鼠模型中，鉴定出此前未被报道的与野生型SpCas9处理相关的脱靶突变。为降低CRISPR-Cas9的脱靶风险，本研究通过生物信息学搜索，鉴定出一种属于II-B亚家族的高固有保真度Cas9变体——副萨特氏菌Cas9（Parasutterella secunda Cas9，PsCas9）。相较于SpCas9，PsCas9在包括PCSK9位点在内的多个检测位点上均表现出更优的特异性，相关实验覆盖体外与体内两种体系。因此，PsCas9可作为SpCas9的替代方案，用于科研与临床应用。我们相信，在未来，双工测序流程的应用将实现对CRISPR-Cas9编辑结果的更灵敏评估，而PsCas9也将为科研与临床应用提供一款更为精准的基因组编辑工具。