Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mol Cell. 2020 Jul 16;79(2):221-233.e5. doi: 10.1016/j.molcel.2020.06.014. Epub 2020 Jun 29.
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 RNA 指导的 DNA 内切酶，在体外能紧密结合底物，但在用于真核细胞基因组操作时会迅速失活。目前尚不清楚是什么因素导致 Cas9 脱落，以及这些因素如何影响基因组工程。通过无偏检测细胞游离的非洲爪蟾卵提取物中的瞬时蛋白相互作用的临近标记，鉴定出二聚体组蛋白伴侣促进染色质转录（FACT）是底物结合的 Cas9 的相互作用因子。FACT 既必需又充分，可以取代 dCas9，并且 FACT 免疫耗竭将 Cas9 的活性从多轮转变为单轮。在人细胞中，FACT 耗竭会延长 dCas9 的停留时间，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 敲低还会增加基于 dCas9 的转录效应物的表观遗传标记，同时增强转录调节。因此，FACT 很可能通过确定 Cas9 的停留时间来塑造细胞对基于 Cas9 的基因组操作的固有反应。
