Matsumoto Daisuke, Matsugi Erina, Kishi Kanae, Inoue Yuto, Nigorikawa Kiyomi, Nomura Wataru
Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
School of Pharmaceutical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Mol Ther Nucleic Acids. 2024 Jan 23;35(1):102124. doi: 10.1016/j.omtn.2024.102124. eCollection 2024 Mar 12.
In genome editing, it is important to avoid off-target mutations so as to reduce unexpected side effects, especially for therapeutic applications. Recently, several high-fidelity versions of SpCas9 have been developed to reduce off-target mutations. In addition to reducing off-target effects, highly efficient intended target gene correction is also essential to rescue protein functions that have been disrupted by single nucleotide polymorphisms. Homology-directed repair (HDR) corrects genes precisely using a DNA template. Our recent development of cell cycle-dependent genome editing has shown that regulation of Cas9 activation with an anti-CRISPR-Cdt1 fusion protein increases HDR efficiency and reduces off-target effects. In this study, to apply high-fidelity SpCas9 variants to cell cycle-dependent genome editing, we evaluated anti-CRISPR inhibition of high-fidelity SpCas9s. In addition, HDR efficiency of high-fidelity SpCas9s was addressed, identifying eSpCas9, SpCas9-HF1, and LZ3 Cas9 as promising candidates. Although eSpCas9 and LZ3 Cas9 showed decreased HDR efficiency in cell cycle-dependent genome editing, SpCas9-HF1 successfully achieved increased HDR efficiency and few off-target effects when co-expressed with an AcrIIA4-Cdt1 fusion.
在基因组编辑中，避免脱靶突变以减少意外的副作用非常重要，尤其是对于治疗应用而言。最近，已开发出几种高保真版本的SpCas9以减少脱靶突变。除了减少脱靶效应外，高效的靶向基因校正对于挽救因单核苷酸多态性而被破坏的蛋白质功能也至关重要。同源定向修复（HDR）使用DNA模板精确校正基因。我们最近开发的细胞周期依赖性基因组编辑表明，用抗CRISPR-Cdt1融合蛋白调节Cas9激活可提高HDR效率并减少脱靶效应。在本研究中，为了将高保真SpCas9变体应用于细胞周期依赖性基因组编辑，我们评估了抗CRISPR对高保真SpCas9的抑制作用。此外，还探讨了高保真SpCas9的HDR效率，确定eSpCas9、SpCas9-HF1和LZ3 Cas9为有前景的候选者。尽管eSpCas9和LZ3 Cas9在细胞周期依赖性基因组编辑中显示出HDR效率降低，但当与AcrIIA4-Cdt1融合蛋白共表达时，SpCas9-HF1成功实现了HDR效率的提高且脱靶效应较少。
