The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.
The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.
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DOI：
10.1016/j.molcel.2020.06.014
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发表时间：
2020-06
期刊：
影响因子：
16
通讯作者：
Alan S. Wang;Leo C. Chen;R. A. Wu;R. A. Wu;Yvonne Hao;D. McSwiggen;Alec B Heckert;Christopher D. Richardson;Benjamin G. Gowen;Katelynn R. Kazane;J. Vu;S. Wyman;Jiyung J. Shin;X. Darzacq;Johannes C. Walter;Johannes C. Walter;Jacob E. Corn;Jacob E. Corn
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文献类型：
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作者：
Alan S. Wang;Leo C. Chen;R. A. Wu;R. A. Wu;Yvonne Hao;D. McSwiggen;Alec B Heckert;Christopher D. Richardson;Benjamin G. Gowen;Katelynn R. Kazane;J. Vu;S. Wyman;Jiyung J. Shin;X. Darzacq;Johannes C. Walter;Johannes C. Walter;Jacob E. Corn;Jacob E. Corn
Cas9 是一种原核 RNA 引导的 DNA 核酸内切酶，可在体外紧密结合底物，但在用于操纵真核细胞中的基因组时会快速翻转。对于导致 Cas9 移位的因素或它们如何影响基因组工程，人们知之甚少。通过对无细胞爪蟾提取物中的瞬时蛋白相互作用进行邻近标记进行无偏检测，鉴定出二聚体组蛋白伴侣促进染色质转录 (FACT) 作为底物结合 Cas9 的相互作用子。 FACT 对于取代 dCas9 来说是必要且充分的，并且 FACT 免疫耗竭将 Cas9 的活性从多周转转化为单周转。在人类细胞中，FACT 缺失会延长 dCas9 的停留时间，延迟基因组编辑，并改变 indel 形成和同源定向修复之间的平衡。 FACT 敲低还增加基于 dCas9 的转录效应子的表观遗传标记，同时增强转录调节。因此，FACT 最有可能通过确定 Cas9 停留时间来塑造细胞对基于 Cas9 的基因组操作的内在反应。
Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightlyin vitrobut turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-freeXenopus laevisegg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.