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

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.