Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Abstract

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. Targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

Figure 1

Architecture of the S. pyogenes CRISPR-Cas9 bound to a guide RNA and a target DNA (4UN3.pdb), with close-up view of the PAM recognition region. Two stars highlight the cleavage sites.

Figure 2

(A) Distribution of conformations in eight independent MD runs (each of ∼1.5 μs) for Cas9-wPAM (gray-to-green) and Cas9-w/oPAM (gray-to-violet), along the principal components of motion (PC1 vs PC2, see also Figure S3). (B) “Open-to-close” conformational transition of Cas9 induced by PAM. (C) GC_ij matrix for Cas9-wPAM (lower triangle) and w/oPAM (upper triangle). GC_ij are color-coded green (correlated) to violet (not correlated). (D) Probability distribution of the GC_ij coefficients wPAM (green) and w/oPAM (violet).

Figure 3

(A) CNA of Cas9–w/oPAM (top) and wPAM (bottom), shown in a 2D representation of the community network. Bonds connect communities and measure their intercommunication strength. (B) GC_ij increased (green) and decreased (magenta) by PAM are plotted on the 3D structure (top), indicating an increase of intercorrelations involving the HNH domain. A per-domain correlation score (Cs_i) matrix (bottom) measures the interdomain correlations wPAM (lower triangle) and w/oPAM (upper triangle). Two boxes highlight the HNH–RuvC intercorrelation. (C) Interactions between essential edges of the allosteric pathway (Q771–K775 and E584–R905), involving the L1 and L2 loops (magenta), which interconnect HNH (green) and RuvC (blue). (D) Electrostatic properties of Cas9–wPAM. Field lines indicate the direction of the electrostatic field from positive (blue) to negative (red) regions. Field lines are filtered according to a gradient magnitude >4.9 kT/e/Å and length >13.5 Å.