Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

Kallimasioti-Pazi, Eirini M.; Chathoth, Keerthi Thelakkad; Taylor, Gillian C.A.; Meynert, Alison; Ballinger, Tracy; Kelder, Martijn J. E.; Lalevée, Sébastien; Sanli, Ildem; Feil, Robert; Wood, Andrew J.

doi:10.1371/journal.pbio.2005595

Cited by 80 publications

(75 citation statements)

References 61 publications

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“…Interestingly, the performance of ScCas9 nuclease is locus-dependent. It has previously been reported that SpCas9 is sensitive to chromatin state, DNA and/ or histone modifications at the target region (Kallimasioti-Pazi et al, 2018). Therefore, we presume that ScCas9 might be more sensitive than SpCas9 to these factors.…”

mentioning

confidence: 76%

Targeted base editing in rice with CRISPR/ScCas9 system

Wang

Gosavi

et al. 2020

Plant Biotechnology Journal

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confidence: 76%

Targeted base editing in rice with CRISPR/ScCas9 system

Wang

Gosavi

et al. 2020

Plant Biotechnology Journal

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“…Additional systematic profiling of repair outcomes, in different compartments (e.g. open versus closed chromatin) and in different sequence contexts, will be necessary to understand the magnitude and nature of each of these potential sources of chromatin-mediated bias (33,39)…”

Section: Discussionmentioning

confidence: 99%

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Chen

McKenna

Schreiber

et al. 2019

Nucleic Acids Research

158

184

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Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.

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“…Programmable prokaryotic nucleases such as Cas9 are widely used for eukaryotic genome and transcriptome manipulation, but it is largely unclear how host cells interface with these foreign enzymes. While several studies have uncovered how histones impede Cas9 target search and binding (Hilton et al, 2015; Horlbeck et al, 2016; Kallimasioti-Pazi et al, 2018; Knight et al, 2015; Yarrington et al, 2018), none have described how proteins responsible for restructuring and remodeling nucleosomes might affect Cas9. We have found that the histone chaperone FACT is required for Cas9 unloading and multi-turnover activity in cell-free extract.…”

Section: Discussionmentioning

confidence: 99%

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

Wang

Chen

et al. 2019

Preprint

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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...

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Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

Cited by 80 publications

References 61 publications

Targeted base editing in rice with CRISPR/ScCas9 system

Targeted base editing in rice with CRISPR/ScCas9 system

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

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

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