Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but 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-free Xenopus laevis egg 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.

Cas9 是一种原核生物 RNA 引导的 DNA 核酸内切酶，可在体外与底物紧密结合，但在用于操纵真核细胞基因组时会迅速转换。关于导致 Cas9 脱落的因素或它们如何影响基因组工程，我们知之甚少。通过无细胞非洲爪蟾中瞬时蛋白质相互作用的邻近标记进行无偏检测鸡蛋提取物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 的相互作用因子。FACT 是取代 dCas9 的必要且充分条件，FACT 免疫耗竭将 Cas9 的活性从多次转换转变为一次转换。在人类细胞中，FACT 耗尽会延长 dCas9 停留时间，延迟基因组编辑，并改变插入缺失形成和同源定向修复之间的平衡。FACT 敲低还增加了基于 dCas9 的转录效应子的表观遗传标记，同时增强了转录调节。因此，FACT 最有可能通过确定 Cas9 停留时间来塑造对基于 Cas9 的基因组操作的内在细胞反应。