RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration

CRISPR J. 2019 Aug;2(4):209-222. doi: 10.1089/crispr.2019.0013.

Abstract

CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems*
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae / genetics
  • Transposon Resolvases

Substances

  • Recombinant Fusion Proteins
  • Tn3 resolvase
  • Transposon Resolvases
  • CRISPR-Associated Protein 9