The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

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 PCT 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.

Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

Interest in CRISPR/Cas9 remains high level as new applications of the revolutionary gene-editing tool continue to emerge. While key structural and biochemical findings have illuminated major steps in the enzymatic mechanism of Cas9, several important details remain unidentified or poorly characterized that may contribute to known functional limitations. Here we describe the foundation of research that has led to a fundamental understanding of Cas9 and address mechanistic uncertainties that restrict continued development of this gene-editing platform, including specificity for the protospacer adjacent motif, propensity for off-target binding and cleavage, as well as interactions with cellular components during gene editing. Discussion of these topics and considerations should inspire future research to hone this remarkable technology and advance CRISPR/Cas9 to new heights.

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