Journal
GENOME BIOLOGY
Volume 18, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/s13059-017-1355-3
Keywords
CRISPR-Cas; Off-target effect; Engineered Cas9 variants; Hammerhead ribozyme-linked sgRNA
Funding
- Institute for Basic Science (IBS) [R021-D1]
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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.
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