Investigating the Impact of Base Pair Mismatches on Cas13d Cleavage Efficiency Using Molecular Dynamics Simulations

CRISPR-Cas13d enzyme has been transformed to an RNA-mediated tool for editing and manipulating nucleic acids, which has great promise in the field of genetic engineering. However, the presence of mismatches significantly undermines the cleavage efficiency of Cas13d to target RNA. The high sensitivity of Cas13d to base mismatches greatly limits its further application in related research areas such as nucleic acid testing and gene therapy. In this work, molecular dynamics simulations were employed to investigate the molecular mechanism of mismatches abolishing the activity of uncultured Ruminococcus sp. Cas13d (UrCas13d). Simulation results demonstrated that base pair mismatches of the target RNA were able to lead to an unwound opening and distortion of the Spacer:Target-RNA duplex, which enhances its interactions with the Helical-1 and Helical-2 domains of UrCas13d. Compared with the on-target system, the increase of those interactions caused by mismatches in mismatch systems hampered the conformation rearrangement of Helical-1 and Helical-2 to form an active conformation. Remarkably, the conformation rearrangement of the Helical-1 domain in mismatch systems also affects the relative position of residues in the HEPN-1 domain, particularly reflected in the movements of both residues Lys274-Phe292 and residues Asp311-Asn330 to residues Ala298-Asn308. Those movements reduced the steric hindrance effect of residues Lys274-Asn330 between residues Tyr212-Lys250 and residues Arg754-Lys772, which stimulated residues Tyr212-Lys250 moving close to residues Arg754-Lys772 in the HEPN-2 domain. The occurrence of this phenomenon resulted in the catalytic center burying into the hydrophobic interior of the UrCas13d protein, which could prevent the contact between the catalytic residues (R-X₄-H motifs) and the target RNA to decrease the cleavage efficiency of Cas13d protein to target RNA. The MD results reveal that blocking the transition of domains from inactive to active conformations and preventing the contact between R-X₄-H motifs and target RNA are the crucial determinants for mismatches to reduce UrCas13d activity. Those results contribute to providing theoretical support for the molecular mechanism of Cas13d that will stimulate future experimental research aimed at designing novel and efficient Cas13d variants to prevent undesired cleavages by regulating the interaction between nucleic acids and domains.