<br>SpCas9-HF1 通过提高 HDR 效率、减少脱靶效应和插入缺失率来提高细胞周期依赖性基因组编辑的准确性,Molecular Therapy - Nucleic Acids

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SpCas9-HF1 通过提高 HDR 效率、减少脱靶效应和插入缺失率来提高细胞周期依赖性基因组编辑的准确性
Molecular Therapy - Nucleic Acids ( IF 6.5 ) Pub Date : 2024-01-23 , DOI: 10.1016/j.omtn.2024.102124
Daisuke Matsumoto _{1,

2} , Erina Matsugi ₁ , Kanae Kishi ₁ , Yuto Inoue ₂ , Kiyomi Nigorikawa _{1,

2} , Wataru Nomura _{1,

2}

Affiliation

在基因组编辑中，重要的是避免脱靶突变，以减少意外的副作用，特别是对于治疗应用。最近，SpCas9 的几个高保真版本被开发出来，以减少脱靶突变。除了减少脱靶效应外，高效的预期靶基因校正对于挽救被单核苷酸多态性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，我们评估了高保真 SpCas9 的抗 CRISPR 抑制。此外，还解决了高保真 SpCas9 的 HDR 效率问题，将 eSpCas9、SpCas9-HF1 和 LZ3 Cas9 确定为有前途的候选者。尽管 eSpCas9 和 LZ3 Cas9 在细胞周期依赖性基因组编辑中显示 HDR 效率降低，但与 AcrIIA4-Cdt1 融合体共表达时，SpCas9-HF1 成功实现了 HDR 效率的提高和很少的脱靶效应。

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SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates

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.

更新日期：2024-01-23

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