CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

Nat Biotechnol. 2013 Sep;31(9):833-8. doi: 10.1038/nbt.2675. Epub 2013 Aug 1.

Abstract

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics*
  • Deoxyribonuclease I / genetics*
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Models, Genetic
  • Molecular Sequence Data
  • RNA, Guide / genetics*
  • Trans-Activators / genetics*

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • Trans-Activators
  • Deoxyribonuclease I