A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

Daisuke Matsumoto,Hirokazu Tamamura,Wataru Nomura

doi:10.1038/s42003-020-01340-2

Daisuke Matsumoto, Hirokazu Tamamura + Show 1 more

Open Access

https://doi.org/10.1038/s42003-020-01340-2

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Abstract

The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

Highlights

The development of genome editing systems based on the Cas[9] endonuclease has greatly facilitated gene knockouts and targeted genetic alterations
homology-directed repair (HDR) occurs during the S and G2 phases, whereas nonhomologous end joining (NHEJ) operates in all phases of the cell cycle, especially in G114
Monomeric Kusabira-Orange fluorescence protein fused with amino acids residues 30 to 120 of Cdt1(Cdt1(30–120)) has been developed[33]

Summary

Introduction

The development of genome editing systems based on the Cas[9] endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. The anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt[1] that is degraded in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. The combination of SpyCas[9] and AcrIIA4-Cdt[1] is a cell cycle-dependent Cas[9] activation system for accurate and efficient genome editing. The CRISPR-Cas[9] system was originally discovered as part of the bacterial immune system against external DNA from organisms such as bacteriophages and plasmids[1,2,3] It has become the predilected simplified genome editing tool, because it is easier and less expensive to construct various target libraries compared to other editing technologies such as ZFN and TALEN. The system displayed autonomous Cas[9] activity switch dependent on the cell cycle

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