Open AccessSpecificity profiling of CRISPR system reveals greatly enhanced off-target gene editing
Author(s) -
Yao Wang,
Wang Mingrui,
Ting Zheng,
Yingzi Hou,
Pingjing Zhang,
Tao Tang,
Jing Wei,
Quan Du
Publication year - 2020
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/s41598-020-58627-x
Subject(s) - crispr , genome editing , endonuclease , cas9 , biology , genetics , gene , trans activating crrna , computational biology , nucleotide , guide rna , mutation , rna editing , subgenomic mrna , rna
To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a “core” sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of ‘super’ off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.