Investigating the role of chromatin modifications in CRISPR/Cas9 gene editing

Abstract

Precisely positioned nucleosomes and heterochromatin have been shown to impede CRISPR/Cas9 editing efficiency. Conversely, Cas9 can open previously inaccessible regions of DNA, and transcriptionally silent targets can usually be edited without difficulty. In order to address this paradox, we have developed a method that exploits the allele-specific chromatin status of imprinted genes to characterise the impact of chromatin modifications on targeted mutagenesis. Cas9 was targeted to imprinted CpG islands in F1 hybrid mouse embryonic stem cells, and then allele-specific mutation patterns were characterised following high throughput amplicon sequencing. Using this novel system, we discovered that heterochromatin can impede mutagenesis with CRISPR/Cas9, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded when Cas9 exposure was brief and when intracellular expression of Cas9 was low, but the consequences of chromatin modifications were minimal following prolonged exposure. The presence of mismatches between single guide RNA and genomic target sequence disproportionately reduced mutagenesis within heterochromatin for some specific combinations of mismatches. This suggests that Cas9 proofreading activity, and hence off-target mutagenesis, may be impacted by chromatin state and merits further investigation. Lastly, no effects of chromatin modifications on the outcome of DNA repair were detected, with similar efficiencies of homology-directed repair (HDR) and non-homologous end joining (NHEJ) on maternal and paternal alleles. Upon further characterisation we discovered that the majority of types of insertions and deletions generated by NHEJ shared similar frequencies between the two alleles. Combined, my data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR/Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.