DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA.

Abstract:

:To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.

journal_name

Genome Res

journal_title

Genome research

authors

Kim D,Kim JS

doi

10.1101/gr.236620.118

subject

Has Abstract

pub_date

2018-12-01 00:00:00

pages

1894-1900

issue

12

eissn

1088-9051

issn

1549-5469

pii

gr.236620.118

journal_volume

28

pub_type

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