Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

Abstract

The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

Figure 1. Identification and quantification of inactive Cas9.

(a) Three different procedures of our partial pre-incubation experiment. (b,c) Quantification of DNA cleavage efficiencies under different pre-incubating conditions: mixing process (b) or temperature during pre-incubation (c; mean±s.e.m., n≥5). (d) CD spectra of Cas9 without tracrRNA at 25 °C (black) and 37 °C (green). Annealing was performed from 37 °C to 25 °C at a cooling rate of −1 °C min⁻¹ (red dotted line). (e) Kinetic curves of DNA cleavage in the absence (grey) or presence (yellow: Cas9-crRNA, blue: Cas9 alone) of 37 °C pre-incubation. DNA incubation and fluorescence measurement were conducted at 37 °C for all plots in (b,c,e). Error bars in e represent s.d.

Figure 2. Single-molecule FRET analysis for sub-conformation of Cas9:gRNA:DNA.

(a) Scheme for smFRET experiment. Cas9 with gRNAs that consist of Cy5 (acceptor)-labelled crRNA, tracrRNA, binds to Cy3 (donor)-labelled dsDNA. In the FRET measurement, a relatively low concentration of gRNAs (30 nM) compared with cleavage experiments was used to reduce the background fluorescence of Cy5-labelled crRNA. (b) A histogram of the FRET upon binding (we selected the molecules emitting Cy5 signal) to wild-type target DNA exhibits two peaks centred at 0.27 and 0.83, with Gaussian fits (black line). (c) A representative time trajectory representing the short-lived open conformation and zipped conformation exhibits two FRET states. The duration of each conformation is measured as the dwell time (Δt). The trajectory was imaged right after the injection of Cas9:gRNA into a single-molecule chamber with an integration time of 0.03 s. The points of binding and photo-bleaching were indicated by the black arrows. (d) Histograms of FRET upon binding with each PAM-distal mutant exhibit different conformational distribution (with Gaussian fits, black line). The ‘mis-crRNA' represents the sequence containing mismatched bases between target and crRNA in PAM-distal region (from +15 to +20) and the ‘bubble DNA' represents the sequence containing mismatched bases between nontarget and target strand in PAM-distal region (from +15 to +20). In b and d, diagrammatic representations are used for PAM (yellow), protospacer (black), crRNA (blue) and the approximate location of mismatched bases on target DNA (red cross).

Figure 3. Effects of crRNA complementarity on R-loop conformations.

(a) A histogram of the FRET upon binding (we selected the molecules emitting Cy5 signal) to wild-type target DNA with 17-nt crRNA exhibits two peaks centred at 0.29 and 0.76, with Gaussian fits (black line). 17-nt crRNA is illustrated by pink line. (b) Comparison with each average dwell time of E_high and E_low from 20-nt or 17-nt crRNA (from dwell time distributions in Supplementary Figs 5 and 6; mean±s.e.m., n≥3).

Figure 4. Cas9 cleavage activity with various mutated target DNA.

(a) The relative target binding (grey bars) versus the fraction of cleaved product (red bars) for DNA sequences containing various positions of mismatched bases. (mean±s.e.m., n≥5). (b) Histograms of FRET upon binding with each mismatched mutant with Gaussian fits (black line). The ratio of high-FRET states to all DNA-bound states calculated from the FRET histograms (white bars) versus the DNA cleavage efficiency (red bars) for different DNA sequences containing various positions of mismatched bases. The base positions are labelled by numbering from the 5′ end of PAM as shown on top panel.

Figure 5. Scheme of the conformational roles of both tracrRNA and crRNA during Cas9 nuclease activity.

Model for the conformational regulation of gRNAs during target DNA binding and cleavage process by Cas9:gRNA complex.