The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells

• DOI: 10.1016/j.molcel.2020.06.014
• Publication Date: 2020-06-29T14:39:16Z
• Authors: Alan S. Wang; Leo C. Chen; R. Alex Wu; Yvonne Hao; David T. McSwiggen; Alec B. Heckert; Christopher D. Ri...; Benjamin G. Gowen; Katelynn R. Kazane; Jonathan T. Vu; Stacia K. Wyman; Jiyung J. Shin; Xavier Darzacq; Johannes C. Walter; Jacob E. Corn
• Keywords: 0301 basic medicine; Biomedical and clinical sciences; DNA Repair; 1.1 Normal biological development and functioning; CRISPR-Associated Proteins; SSRP1; Medical and Health Sciences; Cell Line; Epigenesis, Genetic; Double-Stranded; CRISPRa; Xenopus laevis; 03 medical and health sciences; Genetic; Underpinning research; CRISPR-Associated Protein 9; Genetics; Animals; Humans; DNA Breaks, Double-Stranded; Cas9; Gene Editing; 0303 health sciences; Genome; Genome, Human; Human Genome; DNA Breaks; High Mobility Group Proteins; Health sciences; CRISPRi; DNA; Biological Sciences; Nucleosomes; DNA-Binding Proteins; Biological sciences; FACT complex; histone chaperone; CRISPR; Gene Knockdown Techniques; SPT16; Biochemistry and Cell Biology; Generic health relevance; Transcriptional Elongation Factors; Epigenesis; Human; Developmental Biology

SummaryCas9 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. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9’s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.