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在具有可诱导Cas9表达的hPSC中优化基因淘汰方法和sgRNA选择

Jie Ni^1,2, Junhao Gong¹, Yuqing Ran³

¹State Key Laboratory of Cardiovascular Disease, Chinese Academy of Medical Sciences, Fuwai Hospital, Shenzhen, 12#, Langshan Road, Nanshan District, Shenzhen, 518057, Guangdong, China.

Scientific reports

|November 18, 2025

概括

这项研究优化了人类多能干细胞 (hPSC) 中的CRISPR/Cas9基因淘汰,实现了高效率,并提供了有效的单导向RNA (sgRNA) 选择的可靠方法. 该系统改进了功能遗传研究.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
干细胞生物学干细胞生物学

背景情况:

在功能丧失研究中,CRISPR/Cas9对人类多能干细胞 (hPSC) 的基因淘汰至关重要.
目前的Cas9系统面临着变量效率和识别有效的单导向RNA (sgRNA) 的挑战.

研究的目的:

在hPSC中开发一个优化的CRISPR/Cas9基因淘汰系统,具有高稳定的效率.
为选择有效的sgRNA和评估预测算法提供实际指导.

主要方法:

产生了一个可诱导多西环素的spCas9表达hPSCs (hPSCs-iCas9) 线.
为优化基因淘汰系统系统地改进参数.
评估了gRNA得分算法,并使用了西方涂抹来识别无效的sgRNAs.

主要成果:

实现了高的插入和删除 (INDEL) 效率:单基因的82-93%,双基因淘汰的>80%.
对于大型DNA片段删除,已经证明高达37.5%的同卵性淘汰效率.
对比算法显示了最准确的sgRNA预测;鉴定了ACE2的无效sgRNA,尽管INDELs很高.

结论:

优化的hPSCs-iCas9系统为hPSCs的高效基因淘汰提供了一个强大的框架.
为可靠的sgRNA选择提供实用见解,这对于精确的基因编辑实验至关重要.
强调了在INDEL分析之外验证sgRNA功能的重要性.

关键词:

基因编辑基因编辑在INDEL的效率效率.参数优化的参数优化在 hPSCs-iCas9中使用.sgRNA选择的选择

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CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...