No abstract

is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but 29 turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about 30 the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a 31 proximity labeling system for unbiased detection of transient protein interactions in cell-free 32 Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor 33 of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 34 unloading and converts Cas9's activity from multi-turnover to single-turnover. In human cells, 35 depletion of FACT delays genome editing and alters the balance between indel formation and 36 homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-37 based transcriptional effectors with concomitant enhancement of transcriptional modulation. 38 FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most 39 likely by determining Cas9 residence times.40 41 42 43 44 45 46 47 48 49 50 51 52acetyltransferases or methyltransferases around a transcription start site (TSS) to manipulate 79 expression of endogenous genes (Gilbert et al., 2013; Hilton et al., 2015). Such transcriptional 80 engineering presumably relies on providing these histone modifiers sufficient time at the TSS to 81 deposit the appropriate epigenetic marks. Conversely, Cas9's utility as a targeted nuclease may 82 be predicated on its removal from the genome because Cas9 itself masks the DSB from cellular 83 repair enzymes (Clarke et al., 2018; Richardson et al., 2016b). Cas9 residence times and 84 unloading might thus play crucial roles in Cas9-based interventions. 85 86 While some Cas9 molecules behave as multi-turnover enzymes (Yourik et al., 2019), the widely 87 used Streptococcus pyogenes Cas9 and dCas9 exhibit extremely stable protein-DNA 88 interactions and possess residence times of over five hours in vitro (Raper et al., 2018; 89 Richardson et al., 2016b; Sternberg et al., 2014). Estimates of residence times in live cells vary 90 (Ma et al., 2016; Shao et al., 2016), but some experiments indicate that S. pyogenes Cas9 stays 91 bound to its target in mammalian cells for as little as five minutes (Knight et al., 2015) and imply 92 that cellular factors promote turnover. The ability to detect resolved genomic edits only a few 93 hours after electroporation of Cas9 RNPs (Kim et al., 2014) further suggests that cells actively 94 remove Cas9 from the genome either purposefully or as a byproduct of normal genome 95 metabolism. 97Prior work has suggested that RNA polymerases can dislodge Cas9 from DNA in vitro when the 98 gRNA anneals to the non-coding DNA strand but not to the coding strand (Clarke et al., 2018). 99Targeting the non-coding strand with a gRNA roughly correlated with increased editing rates in 100 human cells and an increased ability of a bacterially-encoded CRISPR system to fight phage 101 infection. However, t...