Matsumoto Daisuke, Kishi Kanae, Matsugi Erina, Inoue Yuto, Nigorikawa Kiyomi, Nomura Wataru
Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan.
School of Pharmaceutical Sciences, Hiroshima University, Japan.
FEBS Lett. 2023 Apr;597(7):985-994. doi: 10.1002/1873-3468.14608. Epub 2023 Mar 22.
Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.
使用CRISPR-Cas9进行基因组编辑，尤其是用于治疗目的时，应通过同源定向修复（HDR）途径来完成，该途径比其他途径具有更高的精确性。然而，有待解决的问题之一是HDR的基因组编辑效率通常较低。据报道，化脓性链球菌Cas9（SpyCas9）与人Geminin融合（Cas9-Gem）可使HDR效率略有提高。相比之下，我们发现，将与染色质许可和DNA复制因子1（Cdt1）融合的抗CRISPR蛋白（AcrIIA4）用于调控SpyCas9的活性，可显著提高HDR效率并减少脱靶效应。在此，应用了另一种抗CRISPR蛋白AcrIIA5，并且Cas9-Gem与抗CRISPR+Cdt1的联合使用显示出HDR效率的协同增强。该方法可能适用于各种抗CRISPR/CRISPR-Cas组合。
