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

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.

SUBMITTER: Kim S

PROVIDER: S-EPMC5686910 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Kim Sojung S Bae Taegeun T Hwang Jaewoong J Kim Jin-Soo JS

Genome biology 20171115 1

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 ...[more]

PMID: 29141659

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