Fusions of Catalytically Inactive RusA to FokI Nuclease Coupled with PNA Enable Programmable Site-Specific Double-Stranded DNA Breaks

Programmable site-specific nucleases have revolutionized genome editing. However, these systems still face challenges such as guide dependency, delivery issues, and off-target effects. Harnessing the natural functions of structure-guided nucleases offers promising alternatives for generating site-specific double-stranded DNA breaks. Yet, structure-guided nucleases require precise reaction conditions and validation for <i>in vivo</i> applicability. To address these limitations, we developed the PNA-coupled FokI-(d)RusA (PC-FIRA) system. PC-FIRA combines the sequence-specific binding ability of peptide nucleic acids (PNAs) with the catalytic efficiency of FokI nuclease fused to a structurally guided inactive RusA resolvase (FokI-(d)-RusA). This system allows for precise double-stranded DNA breaks without the constraints of existing site-specific nuclease and structure-guided nucleases. Through <i>in vitro</i> optimizations, we achieved high target specificity and cleavage efficiency. This included adjusting the incubation temperature, buffer composition, ion concentration, and cleavage timing. Diverse DNA structures, such as Holliday junctions and linear and circular DNA, were tested, demonstrating the potential activity on different target forms. Further investigation has revealed the PC-FIRA system's capacity for facilitating the precise deletion of large DNA fragments. This can be useful in cloning, large-fragment DNA assembly, and genome engineering, with promising applications in biotechnology, medicine, agriculture, and synthetic biology.