Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

Nat Commun. 2017 Apr 7:8:14958. doi: 10.1038/ncomms14958.

Abstract

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites / genetics
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • DNA / genetics
  • DNA / metabolism
  • DNA Cleavage
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Models, Genetic

Substances

  • Bacterial Proteins
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Cas9 protein, Francisella novicida
  • Endonucleases