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Chromatin accessibility and guide sequence secondary structure affect CRISPR ‐Cas9 gene editing efficiency
Author(s) -
Jensen Kristopher Torp,
Fløe Lasse,
Petersen Trine Skov,
Huang Jinrong,
Xu Fengping,
Bolund Lars,
Luo Yonglun,
Lin Lin
Publication year - 2017
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.12707
Subject(s) - crispr , genome editing , cas9 , guide rna , chromatin , biology , euchromatin , genetics , computational biology , gene , heterochromatin
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐associated protein 9 (CRISPR‐Cas9) systems have emerged as the method of choice for genome editing, but large variations in on‐target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9‐mediated gene editing efficiency for 20 gRNA s targeting 10 genomic loci in HEK 293T cells using both SpCas9 and the eSpC as9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR ‐Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9‐mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design.