多功能且高效的哺乳动物基因组编辑使用Desulfovibrio vulgaris的I-C型CRISPR系统
Pan Li1,2,3, Dingcai Dong1, Fei Gao1
1State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing, 100193, China.
Science China. Life sciences
|August 10, 2024
概括
来自Desulfovibrio vulgaris (Dvu) 的新型CRISPR-Cas系统可以实现精确的哺乳动物基因组编辑. 这种类型I-C系统有效地在细胞和动物中创建删除,替换和基编辑.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 目前的CRISPR-Cas基因组编辑工具主要使用Cas9或Cas12a.
- 与I类CRISPR系统一样,I类CRISPR系统显示出增强特异性的潜力,但在编辑方面发展不足.
- 在哺乳动物系统中存在对多功能和精确基因组编辑工具的需求.
研究的目的:
- 开发和描述一种来自Desulfovibrio vulgaris (Dvu) 的I-C CRISPR系统,用于哺乳动物基因组编辑.
- 为了证明Dvu类型I-C编辑器对精确的基因删除,大片段替换和基础编辑的能力.
- 评估Dvu类型I-C系统在各种哺乳动物细胞系和体内模型中的效率和特异性.
主要方法:
- 优化 Dvu 类型 I-C 编辑复合体,使用配对 PAM-in crRNA 策略.
- 编辑器的应用程序用于通过哺乳动物细胞和初级纤维细胞的同质导向修复来产生精确的删除和大片段替代.
- 调整Dvu-Cascade效应器用于细胞因子基编辑 (Dvu-CBE) 和腺因基编辑 (Dvu-ABE).
- 目标之外的分析来评估系统的特异性.
主要成果:
- 在各种细胞系和猪初级纤维细胞中,在多个位点进行有效和精确的缺失生成.
- 使用编辑猪细胞成功生成转基因克隆小猪.
- 通过同质导向修复实现精确的大片段替换.
- 开发Dvu-CBE和Dvu-ABE系统,以实现高效的C-to-T和A-to-G基替代,而无需双链断裂.
- 通过非目标分析证实高特异性.
结论:
- Dvu I-C CRISPR 系统为哺乳动物基因组编辑提供了一个强大的新工具.
- 该系统表现出高效率和特殊性,用于包括删除,片段替换和基础编辑在内的应用.
- Dvu I-C 类型编辑器在生物医学和农业方面的进步具有重大潜力.
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