在人类细胞中,抗CRISPR介导的CRISPR-Cas9的持续定向进化
Andrew L Sabol1,2, Amanuella A Mengiste1,2, Vedagopuram Sreekanth1,3
1Contributed equally to this work.
bioRxiv : the preprint server for biology
|December 19, 2025
概括
研究人员开发了CRISPR-MACE,这是一种用于直接在人体细胞中进行CRISPR-Cas系统持续定向进化的新方法,可以创建改进的基因组编辑工具.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 基因编辑 基因编辑
背景情况:
- 对于研究和治疗来说,CRISPR-Cas系统工程至关重要.
- 当前的优化方法往往无法复制哺乳动物细胞环境.
- 在人类细胞中缺乏持续指导进化的强有力的策略.
研究的目的:
- 引入CRISPR-MACE (哺乳动物细胞启用腺病毒辅助的持续进化) 用于人类细胞中CRISPR-Cas系统的持续定向进化.
- 产生新的Streptococcus pyogenes Cas9 (SpCas9) 变种,具有改变的DNA结合和增强的抑制剂耐药性.
- 建立哺乳动物细胞内CRISPR-Cas系统持续演化的原则.
主要方法:
- 利用基于病毒的连续进化,与基于抗CRISPR的可调性选择集成.
- 在哺乳动物细胞的进化中采用腺病毒辅助的方法.
- 专注于SpCas9变种的定向进化.
主要成果:
- 产生的SpCas9变体具有增加和减少的DNA结合能力.
- 获得了对SpCas9抑制剂AcrIIA4.4的近1000倍增强的耐药性.
- 确定了一种可复制的Cas9守门员突变,使其能够沿着两个功能轴进行适应性进化.
结论:
- 克里斯普-MACE是人类细胞中基因组向剂的基础技术.
- 证明了对改变DNA结合和抑制剂耐药性的SpCas9的进化能力.
- 已确立的原则和合成电路,用于哺乳动物系统中CRISPR-Cas系统的持续演变.
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