Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, USA; Florida State University, Tallahassee, Florida, USA.
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, USA; and Florida State University, Tallahassee, Florida, USA.
CRISPR J. 2021 Apr;4(2):223-232. doi: 10.1089/crispr.2020.0092.
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 (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the catalytic efficiency by AceCECas9. We further evolved the more widely used Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.
在对 CRISPR-Cas9 分子机制有了广泛了解的基础上，蛋白质工程可以成为一种有效的方法，用于改善 CRISPR-Cas9，使其具有不同于天然形式的理想特性。在这里，我们描述了一种定向蛋白质进化方法，该方法可以通过靶向毒性基因内缩短的原间隔序列来选择催化增强的 CRISPR-Cas9 变体（CECas9）。我们用之前从 Staphylococcus aureus 中鉴定出的 II-C 型 Cas9（AceCas9）来验证该方法的有效性，并通过酶动力学显示 AceCECas9 的催化效率提高了四倍。我们进一步对更广泛使用的 Cas9（SpyCas9）进行了进化，并在人结肠癌细胞中证明了 SpyCECas9 促进同源定向修复的基因插入有明显改善。
