Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo, 101-0062, Japan.
Daisuke Matsumoto, Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, USA.
Commun Biol. 2020 Oct 23;3(1):601. doi: 10.1038/s42003-020-01340-2.
The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.
基于 Cas9 内切酶的基因组编辑系统的发展极大地促进了基因敲除和靶向基因修饰。在临床应用中，精确编辑靶基因而不产生脱靶效应对于防止不良反应至关重要。尽管已经报道了几种方法可以减少与 CRISPR 技术相关的脱靶效应，但这些方法往往表现出较低的编辑效率。因此，仍然需要高效、准确和无害的 CRISPR 技术。抗 CRISPR 蛋白是源自噬菌体的 CRISPR-Cas 系统的天然抑制剂。在这里，抗 CRISPR 蛋白 AcrIIA4 与人类 Cdt1 的 N 端区域融合，该区域在 S 和 G 期（即同源定向修复 (HDR) 占主导地位的细胞周期阶段）特异性降解。SpyCas9 和 AcrIIA4-Cdt1 的共表达不仅提高了 HDR 的频率，还抑制了脱靶效应。因此，SpyCas9 和 AcrIIA4-Cdt1 的组合是一种用于精确和高效基因组编辑的细胞周期依赖性 Cas9 激活系统。
Zotero 插件
