Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (gamma PNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the gamma PNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology. Graphical Abstract