Abstract
CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1–4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5–7. Although Cas9 variants with greater discrimination against mismatches have been designed8–10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.
Highlights
We measured the rates of target strand cleavage by Cas[9] in the presence of contiguous triple nucleotide mismatches at different positions along the gRNA–TS duplex (Extended Data Fig. 1a, Extended Data Table 1)
The distal end of the gRNA–TS duplex was in a linear conformation relative to the protospacer adjacent motif (PAM)-proximal DNA–DNA duplex—a state that differs from previously determined on-target DNA-bound Cas[9] structures that depict a kinked duplex[14,18], this state is reminiscent of early R-loop formation intermediates[19]
This is supported by recent structural analyses of catalytically dead Cas[9] in complex with various R-loop formation intermediates, several of which exhibit linear gRNA–TS duplex conformations that are similar to our linear duplex structures[20]
Summary
We sought to understand how certain mismatches can evade Cas[9] discrimination to allow more efficient Cas[9] activation and DNA cleavage relative to other mismatches. R986 is in the ‘down’ conformation, stabilizing the two magnesium ions as predicted by molecular dynamics simulations[22] (Fig. 3), whereas F916 wedges between the −2 and −3 bases through stacking interactions and positions the −3 position within the RuvC active site These observations are in agreement with previous structural and mutagenesis studies[23,24]. As L1 docks on the minor groove, these interactions are gRNA–TS structure-specific rather than sequence-specific and can only occur when the PAM-distal duplex end is in the kinked conformation This provides a structural basis for our observation that the kinked duplex conformation is an intermediate that precedes Cas[9] activation and DNA cleavage. TS(19) participates in water-mediated hydrogen bonds to Q1027, and TS(20) resumes base-pairing with NTS (Fig. 4, Extended Data Fig. 5) This unusual nucleic acid conformation is stabilized by RuvC and appears to facilitate the binding of this mismatch. HNH extending from L1 and L2 linkers has been removed for clarity and does not interact with this region of the gRNA–TS duplex
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