Cell cycle‐dependent regulation of <scp>CRISPR</scp> ‐Cas9 repetitive activation by anti‐ <scp>CRISPR</scp> and Cdt1 fusion in the <scp>CRISPR</scp> a system

CRISPR-Cas9 is a widely used genome-editing tool. We previously developed a method with improved homology-directed repair efficiency and reduced off-target effects by utilizing a fusion protein of AcrIIA4, a Cas9 inhibitor, and Cdt1, which accumulates in the G1 phase and activates Cas9 only in the S/G2 phase. However, it is unknown whether Cas9 inhibition by AcrIIA4 + Cdt1 occurs repeatedly in the G1 phase as the cell cycle progresses. In this study, we used the CRISPRa system to monitor changes in the interaction between Cas9 and AcrIIA4 + Cdt1 at single-cell resolution and in real time. Our findings are among the few examples of successful detection of fluctuating protein-protein interactions that oscillate over time.