Grant: Alliance Grants

Gene editing with RNA-guided nucleases based on the CRISPR bacterial defence system (clustered regularly interspaced palindromic repeats) has revolutionized both basic science and therapeutic applications. However, CRISPR associated (Cas) nucleases are not optimized for all types of gene-editing applications. In particular, the double-strand breaks made in DNA by the Cas proteins are imperfectly repaired by cellular DNA repair pathways that lead to a spectrum of gene-editing outcomes, many of which are undesirable and require downstream experimental identification and validation. The goal of the partnership between Western University and Specific Biologics Inc. is to develop new dual-cleaving RNA guided nucleases that generate defined gene-editing outcomes and that can target sequences not accessible by commonly used Cas9 nucleases, such as SpCas9 (from Streptococcus pyogenes) or SaCas9 (from Staphylococcus aureus). The dual nucleases are based on the TevCas9 nuclease previously developed by the Edgell lab at Western University that is a fusion of the nuclease and linker domains from the I-TevI homing endonuclease to SpCas9 or SaCas9. Given the wide-spread adoption of gene-editing technology by the Canadian academic and biotechnology communities, the new dual-cleaving nucleases developed in this project would provide alternative and improved gene-editing tools for basic scientific studies as well as applications in the biotechnology sector. Such applications include targeted genome modifications to study the effects of gene knockouts in cells and animal models, modification or tagging of therapeutically relevant genes in disease models, and other applications where predictable gene-editing outcomes are required. Proof-of-concept data generated in this partnership would support future downstream therapeutic use of dual-cleaving nucleases to target genetic mutations that cannot be targeted with current gene editors.