Rescue of high-specificity Cas9 variants using sgRNAs with matched 5 ' nucleotides
- Kim, Sojung; Bae, Taegeun; Hwang, Jaewoong; Kim, Jin-Soo
- Issue Date
- BioMed Central
- Genome Biology, Vol.18, p. 218
- 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.