Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Nucleic Acids Res. 2021 Jan 25;49(2):954-968. doi: 10.1093/nar/gkaa1241.
Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.
催化失活的 Cas9（dCas9）已成为一种越来越受欢迎的工具，可用于靶向基因激活/失活、活细胞成像和碱基编辑。尽管已有报道称 dCas9 可诱导碱基替换和缺失，但它与结构变异无关。在这里，我们表明 dCas9 会阻碍复制叉的前进，从而使出芽酵母中的串联重复不稳定。当靶向包含约 16 个重复单元的 CUP1 阵列时，dCas9 在大多数细胞中诱导其收缩，尤其是在存在烟酰胺的情况下。复制中间体分析表明，复制叉在 dCas9 结合位点附近停滞。遗传分析表明，虽然解旋酶复合物成分 Ctf4 和 Mrc1 以及辅助解旋酶 Rrm3 可抵消解旋作用，但它涉及重组蛋白 Rad52 和 Rad59 的单链退火。虽然 dCas9 介导的复制叉停滞在传统应用中是一种潜在的风险，但它可能成为研究机制和操纵基因组不稳定性的新工具。
