Target DNA mutagenesis-based fluorescence assessment of off-target activity of the CRISPR-Cas9 system

The RNA-guided CRISPR/Cas9 system could cleave double-stranded DNA at the on-target sites but also induce off-target mutations in unexpected genomic regions. The base-pairing interaction of sgRNA with off-target DNA was still not well understood and also lacked a direct cell-based assay. Herein we d...

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Bibliographic Details
Published inRSC advances Vol. 9; no. 16; pp. 9067 - 9074
Main Authors Wang, Dan, Niu, Cuili, Han, Jingxin, Ma, Dejun, Xi, Zhen
Format Journal Article
LanguageEnglish
Published England Royal Society of Chemistry 19.03.2019
The Royal Society of Chemistry
Subjects
Assaying
Chemistry
CRISPR
Deoxyribonucleic acid
DNA
Fluorescence
Mutagenesis
Mutation
Nuclease
Ribonucleic acid
RNA
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Summary:The RNA-guided CRISPR/Cas9 system could cleave double-stranded DNA at the on-target sites but also induce off-target mutations in unexpected genomic regions. The base-pairing interaction of sgRNA with off-target DNA was still not well understood and also lacked a direct cell-based assay. Herein we developed a fast target DNA mutagenesis-based fluorescence assay to directly detect the Cas9 activity at different off-target sites in living cells. The results showed that Cas9 nuclease had low tolerance to the nucleotide mismatches in the binding region adjacent to PAM sites, and a tradeoff between Cas9 activity and specificity was also observed compared with the high-fidelity Cas9 variant. The combination of computer-based predictions and this target DNA mutagenesis-based fluorescence assay could further provide accurate off-target prediction guidance to minimize off-target effects to enable safer genome engineering.
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ISSN:2046-2069
2046-2069
DOI:10.1039/c8ra10017a