New RNA-guided dual nucleases for gene editing

基于CRISPR细菌防御系统(成簇的规则间隔的回文重复序列)的RNA引导核酸酶的基因编辑已经彻底改变了基础科学和治疗应用。然而，CRISPR相关(CAS)核酸酶并不是针对所有类型的基因编辑应用而优化的。特别是，由Cas蛋白在DNA中造成的双链断裂不完全被细胞DNA修复途径修复，这导致了一系列基因编辑结果，其中许多是不受欢迎的，需要下游的实验鉴定和验证。西方大学和特定生物公司合作的目标是开发新的双裂解RNA引导核酸酶，这种核酸酶可以产生明确的基因编辑结果，并可以针对常用的Cas9核酸酶无法获得的序列，例如SpCas9(来自化脓链球菌)或SaCas9(来自金黄色葡萄球菌)。双核酸酶是基于之前由西方大学Edgell实验室开发的TevCas9核酸酶，它是从I-Tevi归巢内切酶到SpCas9或SaCas9的核酸酶和连接器域的融合。鉴于加拿大学术界和生物技术界广泛采用基因编辑技术，该项目开发的新的双裂解核酸酶将为基础科学研究和生物技术部门的应用提供替代和改进的基因编辑工具。这些应用包括有针对性的基因组修改以研究细胞和动物模型中基因敲除的影响，疾病模型中治疗相关基因的修改或标记，以及其他需要可预测的基因编辑结果的应用。在这一伙伴关系中产生的概念验证数据将支持未来对双裂解核酸酶的下游治疗使用，以靶向目前的基因编辑无法瞄准的基因突变。

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.