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, 5 (4), 651-662

Deciphering Off-Target Effects in CRISPR-Cas9 Through Accelerated Molecular Dynamics

Affiliations

Deciphering Off-Target Effects in CRISPR-Cas9 Through Accelerated Molecular Dynamics

Clarisse G Ricci et al. ACS Cent Sci.

Abstract

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations-using Gaussian accelerated MD (GaMD)-are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a "lock" effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to "lock" the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to "lock" the catalytic HNH domain in an inactive "conformational checkpoint" is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the "locking" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Conflict of interest statement

The authors declare the following competing financial interest(s): J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics and Mammoth Biosciences, and a Director of Johnson & Johnson. J.A.D. is a scientific advisor to Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences and Inari. J.A.D. has research projects sponsored by Biogen and Pfizer.

Figures

Figure 1
Figure 1
(A) Crystal structure of the S. pyogenes CRISPR-Cas9 system, including the endonuclease Cas9, a guide RNA (orange), the target DNA (TS, cyan), and nontarget DNA (NTS, violet) strands. Cas9 is shown in molecular surface, with protein domains in different colors. The X-ray structure captures the inactive state of the HNH domain, which is a “conformational checkpoint” between DNA binding and cleavage. The right panel highlights the PAM distal sites on the RNA:DNA hybrid and the conformational change of the HNH domain required for catalysis, which is shown with an arrow, indicating the movement of catalytic H840 toward the cleavage site on the TS. (B) Diagram of the DNA and RNA filaments in CRISPR-Cas9, showing the location of base pair mismatches (mm) associated with off-target effects, at PAM distal sites. The model systems considered for Gaussian accelerated MD (GaMD) simulations include the on-target DNA sequence (on-target) and DNA sequences containing base pair mismatches at PAM distal sites (i.e., mm@20, mm@19–20, mm@18–20, and mm@17–20).
Figure 2
Figure 2
Conformations adopted by the RNA:DNA hybrid along GaMD simulations. Representative snapshots extracted from GaMD simulations of the CRISPR-Cas9 system, including the on-target DNA (A) and base pair mismatches (mm) at different positions of the hybrid: mm@20 (B), mm@19–20 (C), mm@18–20 (D), and mm@17–20 (E). “Productive” systems, which efficiently cleave their DNA substrate at rates similar to the on-target Cas9, are highlighted using cool colors (black for the “on-target” CRISPR-Cas9 and blue for the “off-target” systems), whereas the “unproductive” mm@17–20 system, which slowly cleaves the DNA substrate, is highlighted in red (warm color). The RNA (orange) and the target DNA (TS, cyan) are shown as ribbons. Mismatched bases on the TS are highlighted in magenta. The protein environment is shown as a molecular surface. These configurations are representative of the conformational changes detailed in Figures S3–S5 and in Figure 3.
Figure 3
Figure 3
RNA:DNA geometrical properties along GaMD simulations. (A) The RNA:DNA minor groove width has been computed in the PAM distal region at six different levels (i–vi, from positions 20 to 16 of the TS), which are schematically shown on the 3D structure of the RNA:DNA hybrid. The probability distributions of the RNA:DNA minor groove width at the iii–v levels are shown for the on-target CRISPR-Cas9 and for the systems including base pair mismatches at different positions of the hybrid (i.e., mm@20, mm@19–20, mm@18–20, and mm@17–20). A vertical bar indicates the experimental minor groove width (i.e., ∼11 Å from X-ray crystallography, enlarged by ∼1 Å if NMR data are considered). Full data are reported in Figure S2. (B) Scatter plots of the geometrical base pair descriptors, computed at position 17 of the RNA:DNA hybrid for all studied systems (full data are in Figures S3–S5). Translational (i.e., shear, stretch, and stagger) and angular (i.e., buckle, propeller, and opening) descriptors are expressed in Å and degrees, respectively. The RNA:DNA hybrid is shown on the right for the on-target CRISPR-Cas9 (gray) superposed to the mm@17–20 (red) system.
Figure 4
Figure 4
Quantitative evaluation of the interactions between the RNA:DNA hybrid and the Cas9 protein. The number of tight contacts (i.e., within 4 Å radius) established along the dynamics by the RNA:DNA hybrid with the neighboring residues of the HNH and RuvC domains, as well as with the L2 loop connecting HNH to RuvC at PAM distal ends, has been computed for the simulated systems (i.e., on-target, mm@20, mm@19–20, mm@18–20, and mm@17–20). The polar, apolar, and charged groups of residues have been considered. A cartoon of the mm@17–20 system, highlighting the RNA:DNA hybrid and its interactions with HNH and RuvC, as well as with the L1/L2 loops, is shown (right panel).
Figure 5
Figure 5
Locking interactions between the DNA target strand (TS) and the L2 loop of the HNH domain, which decrease the HNH conformational flexibility in the presence of 4 base pair mismatches at PAM distal sites. (A) Representative snapshot of the mm@17–20 system, showing the interactions established by the RNA:DNA hybrid with the residues of the L2 loop. (B) The time evolution of the interactions established by R904, D911, and S908 and the TS at positions 17 (R904) and 18 (D911 and S908) is reported. Data for the on-target Cas9 (black) are compared to the mm@17–20 system (red).
Figure 6
Figure 6
(A) Number of tight contacts (i.e., within 4 Å radius) established along the dynamics by the RNA:DNA hybrid and the neighboring residues of the REC3 region computed for the simulated systems (i.e., on-target, mm@20, mm@19–20, mm@18–20, and mm@17–20). The polar, apolar, and charged groups of residues have been considered. (B) Representative snapshot from GaMD simulations of the mm@17–20 system, showing the extended opening of the RNA:DNA hybrid and the insertion of the 692–700 α-helix (gray) within the heteroduplex. (C) Snapshot from GaMD of the on-target CRISPR-Cas9, showing a well-behaved RNA:DNA hybrid and the conserved interactions established by Q965, N692, and the DNA TS. The RNA (orange) and the DNA TS (cyan) are shown as ribbons. The 692–700 α-helix (gray) is shown as a cartoon; interacting protein residues are shown as sticks.

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