Real-time observation of Cas9 postcatalytic domain motions

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3′ flap generated by NTS cleavage can be rapidly digested by a 3′ to 5′ single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3′ flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9’s conformation and suggest possible biotechnological applications.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system targets foreign nucleic acids for destruction in bacteria and archaea (1). Among different types of the system, CRISPR-Cas9 from Streptococcus pyogenes has been widely used for genome editing in plant and animal cells (2–5). DNA cleavage by the CRISPR-Cas9 system involves multiple steps (6). The Cas9 enzyme associates with a guide RNA, consisting of a programmable CRISPR RNA (crRNA) and a transactivating RNA (tracrRNA), to form a Cas9 ribonucleoprotein complex (RNP) (7). The DNA substrate of Cas9 RNP contains a protospacer region complementary to the spacer sequence of crRNA, and a protospacer adjacent motif (PAM) (NGG for S. pyogenes Cas9) flanking the protospacer (7). After Cas9 RNP binding, the DNA is directionally unwound from the PAM-proximal region to the PAM-distal region (8–10), and the unwound target strand (TS) is hybridized to the spacer sequence of crRNA. The TS and nontarget strand (NTS) of the DNA are cleaved by the HNH domain (residue 780 to 906) and the RuvC domain (residues 1 to 56, 718 to 765, and 926 to 1,099), respectively (7, 11). Engineering of the active site of the HNH domain or RuvC domain creates NTS nickase (Cas9_dHNH, Cas9 with H840A mutation which cleaves NTS only) or TS nickase (Cas9_dRuvC, Cas9 with D10A mutation which cleaves TS only) (7). The TS and NTS Cas9 nickases have also been used in many genome editing applications to achieve higher editing specificity or avoid generating double-stranded breaks (12–14). Cas9 RNP remains stably bound to the cleaved DNA for hours in vitro (8, 9, 15, 16), likely hindering DNA repair processes in cells (15). Therefore, a characterization of the postcatalysis state of Cas9 and its nickase variants has the potential to provide insights into genome editing mechanisms.Fluorescence resonance energy transfer (FRET) and structural studies have demonstrated the key roles of the HNH domain conformation in Cas9-mediated DNA cleavage (17–24). The HNH domain undergoes large conformational changes from the “undocked” inactive conformations to the “docked” active conformation with respect to its TS substrate upon on-target DNA binding, which represents a conformational activation of the HNH domain preceding DNA cleavage (18, 19). The HNH activation also involves conformational changes of the REC2 domain (residue 167 to 307) and REC3 domain (residue 497 to 713) (25). The HNH domain was not visible in a recent cryo-EM structure of the “product” state Cas9-RNA-DNA complex, suggesting the HNH domain is flexible after DNA cleavage (26). However, the previous FRET study showed that the docked conformation persists after DNA cleavage and the product state HNH domain motions were not observed (except for a special case in which the 3′ flap of the NTS was completely removed after DNA cleavage) (18), possibly because the labeling positions for the FRET donor and acceptor pair were not sensitive to the conformational differences between the docked state and the product state of the HNH domain. In this study, we created new Cas9 FRET constructs with increased sensitivity to small distance changes between the FRET pair and observed postcatalytic HNH domain motions using single-molecule FRET.