Bidirectional Degradation of DNA Cleavage Products Catalyzed by CRISPR/Cas9

Abstract

Since the initial characterization of Streptococcus pyogenes CRISPR/Cas9 as a powerful gene-editing tool, it has been widely accepted that Cas9 generates blunt-ended DNA products by concerted cleavage of the target (tDNA) and non-target (ntDNA) strands three nucleotides away from the protospacer adjacent motif (PAM) by HNH and RuvC nuclease active sites, respectively. Following initial DNA cleavage, RuvC catalyzes 3′→5′ degradation of the ntDNA resulting in DNA products of various lengths. Here, we found that Cas9 selects multiple sites for initial ntDNA cleavage and preferentially generates staggered-ended DNA products containing single-nucleotide 5′-overhangs. We also quantitatively evaluated 3′→5′ post-cleavage trimming (PCT) activity of RuvC to find that ntDNA degradation continues up to the −10 position on the PAM distal DNA product and is kinetically significant when compared to extremely slow DNA product release. We also discovered a previously unidentified 5′→3′ PCT activity of RuvC which can shorten the PAM proximal ntDNA product by precisely one nucleotide with a comparable rate as the 3′→5′ PCT activity. Taken together, our results demonstrate that RuvC-catalyzed post-cleavage trimming ultimately generates DNA fragments with heterogeneous ends following initial DNA cleavage including a PAM proximal fragment with a blunt end and a PAM distal fragment with a staggered-end, 3′-recessed on the ntDNA strand. These kinetic and biochemical findings underline the importance of temporal control of Cas9 during gene-editing experiments and help explain the patterns of nucleotide insertions at sites of Cas9-catalyzed gene modification in vivo. Support or Funding Information Florida State University startup fund and a Pelotonia Idea Research Program Award from The Ohio State University Comprehensive Cancer Center A summary figure of my abstract This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.