UC Berkeley

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 that physically interact with or dislodge Cas9 from DNA and how these factors might influence genome engineering tools. I first attempted to identify Cas9 displacement factors by repurposing Cas9 as a proximity sensor in live human cells. In order to reduce the noise derived from unbound Cas9 molecules, I subsequently used cell-free Xenopus laevis egg extract whose lack of a plasma membrane allowed me to tightly control the ratio of Cas9 to its substrate. Label-free proteomics 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.