Utilizing MD Simulation to Understand the Molecular and Structural Mechanism Underlying the DNA Targeting by the INTEGRATE System

Abstract

Recent research has uncovered the INTEGRATE system, a promising gene-editing tool that integrates transposons into the target DNA without the need for double-strand breaks. In contrast to the CRISPR-Cas9 system, the INTEGRATE system avoids off-target effects caused by unintended double-strand breaks. Studies on the INTEGRATE system have identified key factors involved in RNA-guided DNA transposition, including the formation of a complete R-loop structure as a checkpoint state. However, there may be additional checkpoint states and conformations that have yet to be discovered. Molecular dynamic simulations (MD simulations) can provide a detailed atomic-level understanding of the INTEGRATE system and help identify additional checkpoint states that may not have been observed through experimental methods alone. The initial step in the INTEGRATE system is PAM recognition, and the conformational changes that occur during this process can provide valuable insights into the mechanism of action. Through molecular dynamics simulations, the interactions between Cas8 protein and different PAM sequences were studied to identify the key residues involved in PAM recognition. The results demonstrated the importance of the interaction between Arg246 and Guanine at position (-1) of the target strand for PAM recognition. We observed that the presence of unfavorable interactions in the 5'-AC-3' PAM mutant resulted in the disruption of this interaction and accounted for its 0% integration efficiency. Furthermore, our investigation revealed that PAM sequences not only initiate but also modulate the integration process through an allosteric mechanism that connects the N-terminal domain and the helical bundle of Cas8. This allosteric regulation was observed in all PAMs tested, including those with lower integration v efficiencies, such as 5'-TC-3' and 5'-AC-3'. We also identified the specific Cas8 residues that could be treated as allosteric hotspots. Moreover, the effect of PAM-distal mismatches on RNA-guided DNA transposition was found to vary. Mismatches located at positions 25-28 were found to completely block the transposition process, while mismatches in positions 29-32 could be tolerated. Our results showed that the stable rotation of Cas8-HB is a crucial step in the interaction with TniQ and the start of the DNA transposition process. PAM distal mismatches can alter the structure of the DNA-RNA hybrid and the minor groove's width. The use of network analysis techniques has helped in identifying significant residues in protein networks such as Cas8 and TniQ dimers. Overall, the insights obtained from this study contribute to a more comprehensive understanding of the INTEGRATE system and its potential for improving genome engineering.