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

Genome Biol. 2017 Nov 15;18(1):218. doi: 10.1186/s13059-017-1355-3.


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.

Keywords: CRISPR-Cas; Engineered Cas9 variants; Hammerhead ribozyme-linked sgRNA; Off-target effect.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / metabolism*
  • Gene Editing
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Nucleotides / metabolism*
  • RNA, Catalytic / metabolism
  • RNA, Guide, Kinetoplastida / metabolism*


  • CRISPR-Associated Proteins
  • Nucleotides
  • RNA, Catalytic
  • RNA, Guide
  • hammerhead ribozyme