crispr系统中抗crispr和Cdt1融合对CRISPR-Cas9重复激活的细胞周期依赖性调控

IF 3.5 4区生物学 Q1 Biochemistry, Genetics and Molecular Biology

FEBS Letters Pub Date : 2024-12-30 DOI:10.1002/1873-3468.15090

Kanae Kishi, Kiyomi Nigorikawa, Yuki Hasegawa, Yusaku Ohta, Erina Matsugi, Daisuke Matsumoto, Wataru Nomura

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引用次数: 0

摘要

CRISPR-Cas9是一种广泛使用的基因组编辑工具。我们之前开发了一种方法，利用Cas9抑制剂AcrIIA4和Cdt1的融合蛋白，提高了同源性定向修复效率，减少了脱靶效应，Cdt1在G1期积累，仅在S/G2期激活Cas9。然而，随着细胞周期的进展，是否在G1期反复发生AcrIIA4 + Cdt1对Cas9的抑制尚不清楚。在本研究中，我们使用CRISPRa系统在单细胞分辨率下实时监测Cas9与AcrIIA4 + Cdt1相互作用的变化。我们的发现是成功检测随时间波动的蛋白质-蛋白质相互作用的少数例子之一。

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Cell cycle-dependent regulation of CRISPR-Cas9 repetitive activation by anti-CRISPR and Cdt1 fusion in the CRISPRa 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.

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来源期刊

FEBS Letters 生物-生化与分子生物学

CiteScore

7.00

自引率

2.90%

发文量

303

审稿时长

1.0 months

期刊介绍： FEBS Letters is one of the world''s leading journals in molecular biology and is renowned both for its quality of content and speed of production. Bringing together the most important developments in the molecular biosciences, FEBS Letters provides an international forum for Minireviews, Research Letters and Hypotheses that merit urgent publication.