Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

Abstract

The CRISPR/Cas9 system is a popular genome-editing tool with immense therapeutic potential. It is a simple two-component system (Cas9 protein and RNA) that recognizes the DNA sequence on the basis of RNA:DNA complementarity, and the Cas9 protein catalyzes the double-stranded break in the DNA. In the past decade, near-atomic resolution structures at various stages of the CRISPR/Cas9 DNA editing pathway have been reported along with numerous experimental and computational studies. Such studies have boosted knowledge of the genome-editing mechanism. Despite such advancements, the application of CRISPR/Cas9 in therapeutics is still limited, primarily due to off-target effects. Several studies aim at engineering high-fidelity Cas9 to minimize the off-target effects. Molecular Dynamics (MD) simulations have been an excellent complement to the experimental studies for investigating the mechanism of CRISPR/Cas9 editing in terms of structure, thermodynamics, and kinetics. MD-based studies have uncovered several important molecular aspects of Cas9, such as nucleotide binding, catalytic mechanism, and off-target effects. In this Review, the contribution of MD simulation to understand the CRISPR/Cas9 mechanism has been discussed, preceded by an overview of the history, mechanism, and structural aspects of the CRISPR/Cas9 system. These studies are important for the rational design of highly specific Cas9 and will also be extremely promising for achieving more accurate genome editing in the future.

Figure 1

Timeline of pioneering leads related to CRISPR/Cas discovery.

Figure 2

Schematic representation of the CRISPR-mediated immunity acquired in bacteria through (1) adaptation, integration of foreign DNA as a spacer (S0) in CRISPR loci after leader sequence (L) flanked by direct repeats (R); (2) expression, decoding of CRISPR array into pre-crRNA, Cas protein(s), and tracrRNA followed by subsequent processing of pre-crRNA into crRNA; and (3) interference, Cas9 in complex with crRNA:tracrRNA neutralizes the reinfection. The Cas9:crRNA:tracrRNA complex recognizes the foreign DNA by using PAM sequence and DNA:RNA complementarity and triggers Cas9-catalyzed DNA cleavage (the cleavage site is indicated by black arrows), thus providing immunity against viral reinfection. The target DNA (tDNA) strand is shown in orange, and the nontarget strand is shown in yellow. The PAM sequence is shown in the nontarget DNA (ntDNA) strand. The adaptation, expression, and interference stages are represented with gray, green, and blue arrows, respectively.

Figure 3

Simple overview of CRISPR/Cas classification. Cas proteins (oval) involved in various stages of CRISPR function are represented in different colors: gray (adaptation), green (expression), and blue (interference). The Cas9 protein in the type-II system is the focus of this Review, highlighted with a dark border.

Figure 4

(a) Different domains of Cas9 protein and their lengths are represented with different colors. (b) X-ray structure of Cas9 endonuclease adopted from PDB 5F9R (resolution = 3.4 Å), captured in a precleavage state containing sgRNA (orange) and intact dsDNA (yellow). The structure on the right side is rotated by 180° around the axis of sgRNA. The protein is demonstrated as a transparent surface, while the dsDNA and sgRNA are visualized as cartoons.

Figure 5

Comparison of the X-ray crystal structures of Cas9 in different states: (a) apo Cas9 (PDB ID 4CMQ), (b) the Cas9:sgRNA complex (PDB ID 4ZT0), (c) the Cas9:sgRNA:tDNA complex with the PAM sequence of the nontarget strand (PDB ID 4UN3), and (d) the Cas9:sgRNA:dsDNA complex (PDB ID 5F9R). The domains showing the major conformational changes between two consecutive states are highlighted as opaque cartoons (also indicated by colored arrows), while the domains having similar structures between two consecutive stages of Cas9 are represented as transparent cartoons. The target and nontarget strands of DNA are denoted as tDNA and ntDNA, respectively.

Figure 6

Schematic representation of the structure of the CRISPR/Cas9 R-loop complex and the key interactions in the Cas9:crRNA:tracrRNA:dsDNA complex. The tDNA and ntDNA are represented in yellow and orange, respectively. The spacer, repeats, and tracrRNA regions of sgRNA are represented in red, pink, and yellow, respectively. Key interactions associated with nucleotide binding and catalysis are visualized as stick representations in the circles. Mg²⁺ ions were placed in the RuvC and HNH catalytic sites on the basis of models predicted by Zuo and Liu, 2016, and Zhu et al., 2019, respectively.

Figure 7

Pictorial representation highlighting the important findings from the Molecular Dynamics studies. (a) Targeted Molecular Dynamics (tMD) of Cas9:sgRNA → apoCas9 and Cas8:sgRNA:dsDNA → Cas9:sgRNA:tDNA:incomplete-ntDNA. The arrows depict the conformational changes of REC I–III and the rotation of the HNH domains. (b) Distance between catalytic H840 residue and tDNA cleavage site (scissile phosphate) in the presence and absence of complete ntDNA. (c) Roles of different REC domains of Cas9 in nucleotide recognition and catalysis. (d) PAM facilitates allosteric signaling (K775-Q771 and R905-E584 interactions) and establishes the correlation between the HNH and RuvC domains involving the L1 and L2 loops.

Figure 8

(a) Schematic representation of the effect of base-pairing mismatches on HNH activation and off-target effects. (b) Outline of the effect of reducing nonspecific interactions in altering ntDNA flexibility and Cas9 specificity. (c) Protein mutations that have been reported to alter Cas9 specificity. Asterisks (*) denote mutations that reduce the off-target effect, and the blue “▲” denotes a mutation that increases Cas9’s specificity toward the PAM (NGG) motif.