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

Abstract

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.

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

“…Thus, involvement of other factors in the mechanism should be considered. As shown in our recent report using AcrIIA5-Cdt1 with SpCas9, 12 we also observed that AcrIIA4-Cdt1 with SpCas9-HF1 increased HDR efficiency at all target sites and that AcrIIA5-Cdt1 with SpCas9-HF1 failed at the EMX1 site. Success of SpCas9 activity control by AcrIIA5-Cdt1 may depend on target sequences, especially in relation to their melting temperatures (Tm).…”

“…AcrIIA5-Cdt1 increased HDR efficiency and decreased off-target effects, depending on the target DNA sequence. 12 Moreover, Cdt1-fused anti-CRISPRs have shown synergistic increases of HDR efficiency when used with SpCas9 fused with Geminin 1-110 fragment (SpCas9-Gem), which is degraded in G1 phases. 12 , 13 , 14 Cell cycle-dependent genome editing not only increases HDR efficiency, but also suppresses off-target effects.…”

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

“…miCas9 showed higher efficiency rates (2–3-fold increase) for large-size gene knock-in compared to wtCas9, while significantly decreasing off-target events [ 77 ]. Even though other HDR-fusion motifs, such as CtIP [ 30 ], DN1S [ 31 ], Geminin [ 78 ], mSA [ 79 ], and RAD52 [ 80 ], have also been evaluated, these motifs are larger than Brex27 (~306% and 1308%), resulting in potential challenges when viral vectors are used as carriers for the CRISPR/Cas9 system due to their limited packing capacity [ 81 , 82 ]. Even though some efforts have been made to increase the Cas9 specificity without size changes resulting in several Cas9 variants (see Table 1 ), they have a marginal effect on the knock-in efficiency increase [ 26 , 27 , 33 ].…”

The history of genome editing: advances from the interface of chemistry & biology

“…140 This Cdt1-based anti-CRISPR expression regulation is also applicable to AcrIIA5, and when combined with Cas9-Geminin, it shows a synergetic effect on precise genome editing. 141 Studies utilizing anti-CRISPR for precision genome editing by combining it with light- or chemically inducible methods or nucleic acid-mediated control systems such as aptamer and miRNA will enhance the possibility of anti-CRISPR applications with CRISPR-Cas systems. New anti-CRISPRs against many types of Cas protein have been reported continuously since the appearance of the first anti-CRISPR.…”