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

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...

DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing

“…As a result, the PAM-proximal DSB end is exposed for further processing, whereas the other end may be temporally blocked from further processing by the still bound Cas9 [68]. RNA-polymerase and facilitates chromatin transcription (FACT) can remove Cas9 from the PAM-distal side, but how Cas9 is removed during genome editing remains unclear [68,69]. How the tight binding of Cas9 to the PAM-distal DSB end affects DSB repair pathway choice is also unclear.…”

“…Tight binding of Cas9 on the PAM-distal DSB end may alter repair kinetics and/or mechanism [170]. Factors such as RNApolymerase and the histone chaperone FACT promote Cas9 release and may affect DNA repair outcomes [68,69]. cNHEJ is the major repair pathway to repair ionizing radiation (IR) induced DSBs and MMEJ is less efficient [183].…”

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

“…We also note the observed difference in activity of the MISER constructs between bacterial in vivo repression experiments and the in vitro binding activity using BLI. We speculate that the MISER constructs are inherently defective for binding target DNA, but that sufficiently perturbed dsDNA in bacteria-such as during replication, transcription, or other rearrangements-presents enough opportunity in the form of dynamically un-and under-wound dsDNA, or stretches of single-stranded DNA, to allow the gRNA to anneal to the spacer sequence 52,61 . In addition, abundant or overexpressed proteins, as is the case here, can often achieve concentrations exceeding 1 μM inside E. coli cells, so it is also possible that the overall high abundance of the MISER constructs in the bacterial repression assay is contributing to the binding signal 62 .…”

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

“…We also note the observed difference in activity of the MISER constructs between bacterial in vivo repression experiments and the in vitro binding activity using BLI. We speculate that the MISER constructs are inherently defective for binding target DNA, but that sufficiently perturbed dsDNA in bacteria-such as during replication, transcription, or other rearrangements-presents enough opportunity in the form of dynamically un-and under-wound dsDNA, or stretches of single-stranded DNA, to allow the gRNA to anneal to the spacer sequence 51,60 .…”