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

Daisuke Matsumoto; Erina Matsugi; Kanae Kishi; Yuto Inoue; Kiyomi Nigorikawa; Wataru Nomura

doi:10.1016/j.omtn.2024.102124

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

Mol Ther Nucleic Acids. 2024 Jan 23;35(1):102124. doi: 10.1016/j.omtn.2024.102124. eCollection 2024 Mar 12.

Authors

Daisuke Matsumoto^{1

2}, Erina Matsugi¹, Kanae Kishi¹, Yuto Inoue², Kiyomi Nigorikawa^{1

2}, Wataru Nomura^{1

2}

Affiliations

¹ Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
² School of Pharmaceutical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Abstract

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.

Keywords: CRISPR-Cas9; MT: RNA/DNA Editing; genome editing; homology-directed repair; off-target effects; protein degradation.