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FEBS Lett. 2017 Jul;591(13):1892-1901. doi: 10.1002/1873-3468.12707. Epub 2017 Jun 28.

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

Author information

1
Department of Biomedicine, Aarhus University, Denmark.
2
University of Cambridge, UK.
3
BGI-Shenzhen, China.
4
China National GeneBank-Shenzhen, BGI-Shenzhen, China.
5
Biology Department, Copenhagen University, Denmark.

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.

KEYWORDS:

CRISPR ; gRNA ; chromatin accessibility; efficiency

PMID:
28580607
DOI:
10.1002/1873-3468.12707
[Indexed for MEDLINE]
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