Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden.
Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
Nat Commun. 2023 Sep 6;14(1):5474. doi: 10.1038/s41467-023-41240-7.
Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
化脓性链球菌 Cas9（SpCas9）和衍生酶被广泛用作基因组编辑工具，但它们的非特异性核酸酶活性常常导致不必要的突变和染色体重排。已经出现了几种用于检测脱靶效应的策略，但它们的灵敏度有限。为了提高检测灵敏度，我们开发了一种使用双链测序的脱靶评估工作流程。该策略将灵敏度提高了一个数量级，在人源化 PCSK9 小鼠模型中鉴定出了先前未知的 SpCas9 脱靶突变。为了降低脱靶风险，我们进行了生物信息学搜索，并从 Parasutterella secunda（PsCas9）中鉴定出 II-B 亚家族的高保真 Cas9 变体。与 SpCas9 相比，PsCas9 在多个测试位点（包括 PCSK9 位点）的体外和体内均显示出更高的特异性。在未来，虽然 PsCas9 将为研究和临床应用提供 SpCas9 的替代方案，但双链测序工作流程将能够更敏感地评估 Cas9 编辑结果。
