针对基因组编辑的双pH响应的CRISPR/Cas9核糖蛋白异复合体
Xianjin Luo1, Janin Germer1, Tobias Burghardt1
1Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
概括
新的异种 (XP) 有效地提供CRISPR/Cas9基因编辑工具,增强细胞吸收和基因组编辑. 这些适应pH的载体对治疗诸如杜申肌肉发育不良等遗传疾病有前途.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 基因工程是一种基因工程.
背景情况:
- 聚类定期间隔的短平行体重复 (CRISPR) /CRISPR相关 (Cas) 蛋白质系统是治疗遗传疾病的强大工具.
- 对于CRISPR/Cas9核糖核蛋白 (RNP) 复合体,有效的传递载体是有限的,阻碍了更广泛的应用.
- 双重pH反应的两异 (XPs) 为CRISPR/Cas9 RNP传递提供了一个潜在的解决方案.
研究的目的:
- 评估二重pH响应的两类异 (XPs) 作为CRISPR/Cas9 RNP传递的载体.
- 评估这些XP在细胞吸收,内体破坏和各种细胞系的基因组编辑方面的效率.
- 优化XP结构以提高基因编辑效率,并探索它们在同质导向修复 (HDR) 中的实用性.
主要方法:
- 设计和合成的人工脂-脂 (lipo-XPs),具有不同比例的氨酸脂肪酸 (LAF) 和氨基乙烯酸 (Stp) 单元.
- 在四个记者细胞系中选了功能Cas9/sgRNA RNP传递的Lipo-XPs,包括杜氏肌肉发育不良 (DMD) 模型.
- 将Cas9/sgRNA RNP复合体与Cas9 mRNA/sgRNA复合体进行比较,并对改进的基因编辑进行了测试.
主要成果:
- 类表明显著增强细胞吸收和HeLa细胞的有效内干扰.
- 几种由XP传递的Cas9/sgRNA RNP复合体在低sgRNA度 (5nM) 的记者细胞系中显示出强大的基因组编辑.
- 优化的XP类似物实现了高基因编辑效率 (EC50降至0.51nM),并促进了高达43%的显著同质导向修复 (HDR).
结论:
- 双重pH响应的两性异代表了可调和有效的CRISPR/Cas9 RNP复合体和RNP/捐赠者DNA多重复合体的输送系统.
- 这些载体为基因编辑应用提供了有前途的战略,包括潜在的遗传疾病治疗.
- 开发的外基因系统显示出高效率和适用于各种基因编辑方式的应用性.
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