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Rescue of high-specificity Cas9 variants using sgRNAs with matched 5 ' nucleotides

Cited 24 time in Web of Science Cited 30 time in Scopus
Authors
Kim, Sojung; Bae, Taegeun; Hwang, Jaewoong; Kim, Jin-Soo
Issue Date
2017-11
Publisher
BioMed Central
Citation
Genome Biology, Vol.18, p. 218
Keywords
CRISPR-CasOff-target effectEngineered Cas9 variantsHammerhead ribozyme-linked sgRNA
Abstract
We report that engineered Cas9 variants with improved specificity-eCas9-1.1 and Cas9-HF1-are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5' terminus, relative to target DNA sequences. Because the nucleotide at the 5' end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5' nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.
ISSN
1474-760X
Language
English
URI
http://hdl.handle.net/10371/138400
DOI
https://doi.org/10.1186/s13059-017-1355-3
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College of Natural Sciences (자연과학대학)Dept. of Chemistry (화학부)Journal Papers (저널논문_화학부)
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