The Impact of Mismatches Within the RNA: DNA Hybrid in the Transposon-Encoded Type I-F CRISPR-Cas System

Recent research has revealed a collaboration between type I-F CRISPR systems and Tn7 transposons in certain bacteria, leading to the discovery of a new gene-editing tool called INTEGRATE. This system integrates transposons into the target strand without introducing a double-strand break or repair mechanism, making it highly promising. The published results showed that a full match between the spacer region of crRNA and the target DNA is necessary to form a stable and complete R-loop, which is critical for accurate integration. PAM-distal mismatches affected RNA-guided DNA transposition differently, with mismatches in positions 25-28 completely blocking the process, while mismatches in positions 29-32 were tolerated. To understand the impact of PAM-distal mismatches on the R-loop stabilization and transposition process, classical all-atom molecular dynamics simulations were conducted using three independent models, including one with a complete RNA-DNA match and two with PAM-distal mismatches located at positions 25-32. Our results suggest that the stable rotation of Cas8-HB is a critical step in the interaction with TniQ and initiation of the DNA transposition process. Network analysis techniques were employed to investigate the communication pathways within Cas8 and TniQ dimers, including eigenvector centrality and correlation analysis. These techniques revealed that certain amino acids in TniQ and Cas8 were highly central to the communication pathways, as they exhibited significant changes in centrality under both mismatches. The findings discussed in this study provide valuable insights into the mechanisms involved in the DNA transposition process and shed light on how PAM-distal mismatches can affect these mechanisms.