Talk abstracts

Talk on Wednesday 03:15-03:30pm submitted by Anthony Stephenson

Promiscuity and auxillary end-trimming activity of Sp. CRISPR/Cas9

Anthony A. Stephenson (OSBP), Austin T. Raper (OSBP)

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 demonstrates promiscuity in selection of the initial ntDNA cleavage site and preferentially generates staggered-ended DNA products containing single-nucleotide 5ʹ-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, 3ʹ-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.

Keywords: CRISPRCas9, enzyme kinetics, exonuclease