Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.

中文翻译：

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通过在酵母中的体内筛选鉴定了一种高度特异性的 SpCas9 变体。

尽管 CRISPR-Cas9 核酸酶可用于基因组编辑，但脱靶活性的潜力限制了它​​们的应用，尤其是用于治疗目的。我们开发了一种基于酵母的检测方法来识别优化的化脓性链球菌 Cas9 (SpCas9) 变体，从而能够同时评估上靶和脱靶活性。我们筛选了在 REC3 域中携带随机突变的 SpCas9 变体库，并确定了在保持编辑效率的同时提高编辑准确性的突变。我们结合了四种有益突变来生成 evoCas9，该变体的保真度超过野生型（79 倍改进）和合理设计的 Cas9 变体（平均改进四倍），同时保持接近野生型的靶向编辑效率（90%中值残留活性）。在评估内源基因组位点上的 evoCas9 时，我们展示了显着提高的特异性，并且在测试的八个单向导 RNA (sgRNA) 中的四个没有观察到脱靶位点。最后，我们表明，在长期表达（40 天）后，evoCas9 强烈限制了难以区分的脱靶位点的非特异性切割，并完全消除了另外两个脱靶位点的切割。