The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Abstract

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. 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 Cas9s 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.