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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Published in | RSC advances Vol. 9; no. 16; pp. 9067 - 9074 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
England
Royal Society of Chemistry
19.03.2019
The Royal Society of Chemistry |
Subjects | |
Online Access | Get full text |
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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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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2046-2069 2046-2069 |
DOI: | 10.1039/c8ra10017a |