Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus

Cell Mol Life Sci. 2015 Jan;72(2):383-99. doi: 10.1007/s00018-014-1679-z. Epub 2014 Jul 20.

Abstract

The Streptococcus thermophilus CRISPR3-Cas (StCas9) system has been shown to mediate DNA cleavage in its original host and in E. coli as well as in vitro. Here, we have reconstituted the StCas9 system in yeast and conducted a systematic optimization of the sgRNA structure, including the minimal length of tracrRNA, loop structure, Match II region, Bulge motif, the minimal length of guide sequence within the crRNA, tolerance of mismatches and target sequence preference. The optimal sgRNA design for the StCas9 system achieved up to 12 and 40 % targeting efficiencies in yeast and human cells, respectively. This study provides important insight into the sequence and structural requirements necessary to develop a targeted and highly efficient eukaryotic gene editing platform using CRISPR-Cas systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / physiology*
  • Cloning, Molecular
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA Editing / genetics*
  • RNA, Messenger / genetics*
  • Saccharomyces cerevisiae
  • Streptococcus thermophilus / genetics*

Substances

  • RNA, Messenger