New RNA-guided dual nucleases for gene editing

用于基因编辑的新型RNA引导双核酸酶

• 批准号: 571374-2021
• 负责人: Edgell, DavidDR
• 金额: $ 5.46万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Alliance Grants
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744036
• 关键词: New; RNA; guided; dual; nucleases

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.

基于CRISPR细菌防御系统（成簇的规则间隔回文重复序列）的RNA引导核酸酶的基因编辑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进行优化的。特别是，由Cas蛋白在DNA中造成的双链断裂通过细胞DNA修复途径不完全修复，导致一系列基因编辑结果，其中许多是不希望的，需要下游实验鉴定和验证。西部大学和特定生物制品公司之间的合作伙伴关系的目标。开发新的双切割RNA引导的核酸酶，其产生确定的基因编辑结果，并且可以靶向通常使用的Cas9核酸酶无法接近的序列，例如SpCas 9（来自化脓性链球菌）或SaCas 9（来自金黄色葡萄球菌）。双核酸酶是基于TevCas 9核酸酶先前由西部大学Edgell实验室开发的，其是来自I-TevI归巢核酸内切酶的核酸酶和接头结构域与SpCas 9或SaCas 9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，该项目开发的新型双切割核酸酶将为基础科学研究以及生物技术部门的应用提供替代和改进的基因编辑工具。这些应用包括靶向基因组修饰，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饰或标记，以及其他需要可预测基因编辑结果的应用。该合作伙伴关系中产生的概念验证数据将支持未来双切割核酸酶的下游治疗用途，以靶向当前基因编辑器无法靶向的基因突变。