Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair–based gene insertion in human colon cancer cells.

中文翻译：

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通过定向蛋白质进化催化增强 Cas9

在 CRISPR-Cas9 分子机制的广泛知识指导下，蛋白质工程可以成为改进 CRISPR-Cas9 以获得不同于其天然形式的所需性状的有效方法。在这里，我们描述了一种定向蛋白质进化方法，该方法能够通过靶向有毒基因内缩短的原型间隔区来选择催化增强的 CRISPR-Cas9 变体 (CECas9)。我们利用先前鉴定的来自解纤维热菌的 II-C 型 Cas9 (AceCas9) 证明了该方法的有效性，并通过酶动力学证明AceCECas9 的体外催化效率提高了四倍。我们进一步进化了更广泛使用的化脓性链球菌Cas9 (SpyCas9)，并证明 SpyCECas9 促进的同源定向修复在人类结肠癌细胞中的基因插入有显着改善。