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.