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. [ABSTRACT FROM AUTHOR]

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