The impact of chromatin dynamics on Cas9-mediated genome editing in human cells

Abstract

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits editing at specific target sites, and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

Chromatin accessibility and guide sequence secondary structure affect CRISPR‐Cas9 gene editing efficiency

“…dCas9 binding and SpCas9 nuclease activity have previously been shown to correlate with an absence of repressive histone marks and increased DNase I sensitivity in human cancer cells. This was shown by Chen et al .…”

“….and Daer et al . using inducible model systems, and by Chari et al . using a library‐on‐library approach.…”

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

“…Importantly, all these potential cleavage sites map specifically to various types of ERV sequences, while off‐target sites were not detected in the CHO genome. Although these sgRNA sequences contain a multitude of predicted target sites, we hypothesized that expressed ERVs might be preferentially cleaved by the CRISPR‐Cas9 nuclease, due to the more efficient cleavage of open chromatin (Daer, Cutts, Brafman, & Haynes, ).…”

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…Several authors showed that Cas9 cleavage efficiency positively correlates with open chromatin based on DNase I hypersensitivity. Along the same line, the activity of Cas9 can be significantly hindered by compact heterochromatin in cells [21]. Interestingly, the engineered Cas9 variants designed to improve specificity, Cas9-HF1 [22] and eSpCas9(1.1) [23], might be even more impacted than Cas9 by the chromatin-related factors [24].…”

“…While treatment with chromatin-disrupting drugs does not appear sufficient, transient overexpression of a targeted transcriptional activator might be an effective method to enhance Cas9 editing at closed chromatin regions [21]. …”