Cas9‐Geminin and Cdt1‐fused anti‐CRISPR protein synergistically increase editing accuracy

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...

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Bibliographic Details
Published in:FEBS letters 2023-04, Vol.597 (7), p.985-994
Main Authors: Matsumoto, Daisuke, Kishi, Kanae, Matsugi, Erina, Inoue, Yuto, Nigorikawa, Kiyomi, Nomura, Wataru
Format: Article
Language:English
Subjects:
anti‐CRISPR
cell cycle
Cell Cycle Proteins - genetics
CRISPR-Cas Systems
Geminin - genetics
Gene Editing
genome editing
homology‐directed repair
Humans
off‐target effects
Recombinational DNA Repair
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Summary: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. Accurate genome editing is achieved by increasing the efficiency of homology‐directed repair (HDR) and reducing off‐target effects. The cell‐cycle‐dependent regulation of targeted DNA cleavage is a key challenge. The addition of human Geminin to SpyCas9 further improved the accuracy of genome editing in combination with a fusion of anti‐CRISPR and Cdt1, suggesting the broad applicability of this method to various anti‐CRISPR/CRISPR‐Cas combinations.
ISSN:0014-5793
1873-3468
DOI:10.1002/1873-3468.14608