Ohio State Biochemistry Program , The Ohio State University , Columbus , Ohio 43210 , United States.
The James Comprehensive Cancer Center , The Ohio State University , Columbus , Ohio 43210 , United States.
J Am Chem Soc. 2018 Sep 12;140(36):11142-11152. doi: 10.1021/jacs.8b05469. Epub 2018 Aug 30.
Interest in CRISPR/Cas9 remains high level as new applications of the revolutionary gene-editing tool continue to emerge. While key structural and biochemical findings have illuminated major steps in the enzymatic mechanism of Cas9, several important details remain unidentified or poorly characterized that may contribute to known functional limitations. Here we describe the foundation of research that has led to a fundamental understanding of Cas9 and address mechanistic uncertainties that restrict continued development of this gene-editing platform, including specificity for the protospacer adjacent motif, propensity for off-target binding and cleavage, as well as interactions with cellular components during gene editing. Discussion of these topics and considerations should inspire future research to hone this remarkable technology and advance CRISPR/Cas9 to new heights.
人们对 CRISPR/Cas9 的兴趣依然很高，因为这项革命性的基因编辑工具不断有新的应用出现。虽然关键的结构和生化发现已经阐明了 Cas9 酶促机制的主要步骤，但仍有几个重要的细节尚未确定或描述不足，这些细节可能是导致已知功能限制的原因。在这里，我们描述了导致对 Cas9 有基本了解的研究基础，并解决了限制该基因编辑平台进一步发展的机制不确定性，包括对前导邻近基序的特异性、脱靶结合和切割的倾向，以及在基因编辑过程中与细胞成分的相互作用。讨论这些主题和注意事项应该会激发未来的研究，以完善这项卓越的技术，并推动 CRISPR/Cas9 达到新的高度。
