Dimeric CRISPR RNA-guided FokI Nucleases for Highly Specific Genome Editing

Nat Biotechnol. 2014 Jun;32(6):569-76. doi: 10.1038/nbt.2908. Epub 2014 Apr 25.

Abstract

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Endonucleases / chemistry*
  • Endonucleases / genetics
  • Gene Editing / methods*
  • Humans
  • Protein Multimerization
  • RNA, Guide / genetics*
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Recombinant Fusion Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific

Associated data

  • SRA/SRP041291