Genetic engineering often uses protein enzymes for sequence-specific nucleic acid manipulation, but these enzymes lack customizability. Programmable systems like TALEN and CRISPR/Cas address this but have limitations, such as engineering difficulties and off-target effects. Large protein enzymes may also struggle to access tightly packed chromatin. Catalytic nucleic acid-based systems, like DNAzymes, are smaller, more stable, and cost-effective, and have been used in various applications. However, their activity on double-stranded DNA (dsDNA) has not been verified, limiting their use in direct gene manipulation.

This is a method for genome editing of double-stranded DNA with higher specificity than existing genome editing technologies. As opposed to relying on a site-specific guide RNA and a non-specific enzyme with the CRISPR/Cas9 system, this method uses Peptide Nucleic Acid (PNA) and DNAzyme, where both components are site specific. The PNA binds to a single strand of dsDNA for the DNAzyme to target and cleave the second strand of DNA. This system achieves the optimal conditions necessary for both PNA invasion of the dsDNA and the DNAzyme cleavage activity. With modification to the conditions, the PNA in this system can be replaced with long homologous ssDNA for increased cost-effectiveness. Inventors have also shown the use of an interstrand zipper for dsDNA invasion, instead of PNA (Tech ID 2024-242).

Publication

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity. Mingkuan Lyu, Linggen Kong, Zhenglin Yang, Yuting Wu, Claire E. McGhee, and Yi Lu. Journal of the American Chemical Society 2021 143 (26), 9724-9728 DOI: 10.1021/jacs.1c03129 [2]