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

Kristopher Torp Jensen, Lasse Fløe, Trine Skov Petersen, Jinrong Huang, Fengping Xu, Lars Bolund, Yonglun Luo, Lin Lin

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Abstract

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 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(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.

Original languageEnglish
JournalFEBS Letters
Volume591
Issue13
Pages (from-to)1892-1901
Number of pages10
ISSN0014-5793
DOIs
Publication statusPublished - 5 Jun 2017

Keywords

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