一个工程核酶编辑器,用于增强向基因组集成
bioRxiv : the preprint server for biology
|September 11, 2023
概括
我们开发了enGager,这是一种新的系统,通过改善DNA模板到目标站点的传递来增强基因编辑. 这种系统显著提高了大型转基因整合的效率,特别是在初级细胞中,为先进的基因疗法铺平了道路.
科学领域:
- 分子生物学分子生物学
- 基因编辑技术的技术
- 细胞工程 细胞工程
背景情况:
- 非病毒性DNA供体模板对于同源重组 (HR) 介导的向基因组集成至关重要.
- 克里斯普尔/Cas9系统已经提高了人力资源的效率,但在DNA供体贩运和大型转基因的局部度方面仍然存在挑战.
- 圆形单链DNA (cssDNA) 已经显示出作为基因组工程催化剂 (GATALYST) 的承诺,但面临着大型插入的局限性.
研究的目的:
- 开发一个增强的基因组工程系统 (enGager),以克服核等离子体贩运和ssDNA捐赠者的基因组绑定的局限性.
- 提高向基因敲门的效率,特别是对于大型转基因,使用非病毒传递方法.
- 为先进的细胞和基因治疗应用创建新的三方编辑器 (TESOGENASETM).
主要方法:
- 通过将核定位信号 (NLS) 标记的Cas9与单链DNA (ssDNA) 结合蛋白质模块融合,开发了enGager系统.
- 利用GFP报告员的敲门选来识别和优化增强器件的组件.
- 组装了工程核酶,sgRNA和ssDNA捐赠者的三方复合体,以增强基因组集成.
- 测试了用于大型转基因 (例如,CAR) 集成在各种细胞类型,包括初级T细胞的增大器系统.
主要成果:
- 与未融合编辑器相比,enGager系统显著提高了针对性基因组集成效率1.5至6倍以上.
- 对于大于4Kb的转基因来说,增强更为明显,特别是在初级细胞中.
- enGager介导的增强依赖于ssDNA捐赠者,但对双链DNA (dsDNA) 的依赖较小.
- 通过使用迷你增大剂,在初级T细胞中证明了大型仿真抗原受体 (CAR) 转基因的高效集成,从而产生功能性抗瘤活性.
结论:
- 形成三方复合体的enGager系统有效地促进了DNA捐赠者的贩运,并增加了目标地点的局部度.
- TESOGENASETM编辑器代表了SSDNA介导的非病毒基因组工程的新工具集.
- 这项技术在开发先进的细胞和基因疗法方面具有显著的潜力,以高效的CAR-T细胞工程为例.
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