PAM-readID是一种快速,简单和准确的PAM测定方法,用于哺乳动物细胞中的CRISPR-Cas酶
Qiuyan Wang1, Zhuang Wang2, Huiyu Liu3
1Zhejiang Key Laboratory of Medical Epigenetics, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, China. qywang@hznu.edu.cn.
Communications biology
|November 18, 2025
概括
一种名为PAM-readID的新方法可以准确地确定哺乳动物细胞中CRISPR-Cas核酶原空间器相邻基因 (PAM) 配置文件. 这种快速的技术简化了PAM识别,推进了基因组编辑应用.
科学领域:
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
- 生物技术是生物技术.
背景情况:
- 在CRISPR-Cas核酶原空间体相邻基因 (PAM) 配置文件在实验系统 (体外,细菌,哺乳动物细胞) 之间有所不同.
- 哺乳动物细胞中现有的PAM测定方法复杂,阻碍了广泛采用.
- 对于专门针对哺乳动物系统的强大的PAM定义方法有着至关重要的需求.
研究的目的:
- 开发一种快速,简单和准确的方法来确定哺乳动物细胞中的CRISPR-Cas核酶PAM识别配置文件.
- 为PAM分析建立一个高通量测序的经济有效的替代方案.
主要方法:
- 引入了PAM-readID (通过DNA双链断裂中的双链寡氧核酸集成实现的PAM识别-配置-确定).
- 使用PAM-readID分析哺乳动物细胞中多个CRISPR-Cas核酶 (SaCas9,SaHyCas9,Nme1Cas9,SpCas9,SpG,SpRY,AsCas12a) 的PAM概况.
- 使用低序列深度分析和桑格测序验证了PAM-readID的准确性.
主要成果:
- 在哺乳动物细胞中成功生成了几个CRISPR-Cas核酶的全面PAM识别配置文件.
- 证明了精确的SpCas9 PAM偏好识别,只需要500次序读取.
- 展示了PAM-readID通过桑格测序来确定Cas9 PAM配置文件的能力,与高吞吐量方法相比,可以节省大量的时间和成本.
结论:
- PAM-readID提供了一个可访问和有效的工具,用于表征哺乳动物细胞中CRISPR-Cas核酶的功能PAM.
- 这种方法促进了新型基因组编辑核酶的发现和应用.
- 加快了用于哺乳动物基因组工程的CRISPR-Cas技术的进展.
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