DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Figure 1. DNA curtains assay for target binding by Cas9:RNA

a, Schematic of a single-tethered DNA curtain^,. b, Wild-type Cas9 or dCas9 was programmed with crRNA:tracrRNA targeting one of six sites. c, YOYO1-stained DNA (green) bound by QD-tagged dCas9 (magenta) programmed with λ2 guide RNA. d, dCas9:RNA binding distributions; error bars represent 95% confidence intervals obtained through bootstrap analysis. e, Image of apo-Cas9 bound to DNA curtains bound to apo-Cas9. f, Binding distribution of apo-Cas9; error bars represent 95% confidence intervals. g, Lifetimes of DNA-bound apo-Cas9 and Cas9:RNA after injection of λ2 crRNA:tracrRNA (100 nM) or heparin (10 μg mL⁻¹).

Figure 2. Cas9:RNA remains bound to cleaved products and localizes to PAM-rich regions during the target search

a, Wild-type Cas9:RNA bound to DNA curtains. b, Cleavage yield of 25 nM plasmid DNA is proportional to [Cas9:RNA]. c, Schematic of a double-tethered DNA curtain^-. d, Liberation of the cleaved DNA with 7 M urea; asterisks denote QDs that are attached to the lipid bilayer but not bound to the DNA. e, Kymographs illustrating distinct binding events. f, Survival probabilities for non-target binding events; solid lines represent double-exponential fits. Inset: survival probabilities of DNA-bound apo-Cas9 and target DNA-bound Cas9:RNA. g, Distribution of Cas9:RNA binding events (N = 2,330) and PAM density. Colour-coding reflects the binding dwell time (t_i) relative to the mean dwell time (t̄). h, Correlation of PAM distribution and non-target Cas9:RNA binding for λ2 (blue) and spacer 2 (green) guide RNAs.

Figure 3. Cas9:RNA searches for PAMs and unwinds dsDNA in a directional manner

a, Schematic of the competition cleavage assay. b, Cleavage assay with and without competitor DNAs. c, Quantitation of competition data (mean ± s.d.). Competitor cartoon representations show PAMs (yellow) and regions complementary to the crRNA (red). d, Predicted data trends for the random nucleation or sequential unwinding models aligned with the corresponding data in (e). e, Competition assays using substrates with variable degrees of crRNA complementarity, shown as in (c). Numeric descriptions of the competitor DNAs indicate the regions of complementarity (red) or mismatches (black) to the crRNA sequence.

Figure 4. PAM recognition regulates Cas9 nuclease activity

a, Cleavage assay with single-stranded, double-stranded, and partially double-stranded substrates. b, Relative affinities and cleavage rates; (mean ± s.d.). c, Model for target search, recognition and cleavage by Cas9:RNA. The search initiates through random 3D collisions. Cas9:RNA rapidly dissociates from non-PAM DNA, but binds PAMs for longer times and samples adjacent DNA for guide RNA complementarity, giving rise to a heterogeneous population of intermediates. At correct targets, Cas9:RNA initiates formation of an RNA:DNA heteroduplex, and R-loop expansion propagates via sequential unwinding. The DNA is cleaved and Cas9:RNA remains bound to the cleaved products.