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一个紧的CRISPR-Cas效应器Cas12h1的分子洞察力和理性工程与一个宽频的PAM

Weiwei Zheng¹, Hongyu Li¹, Mengxi Liu¹

¹Key Laboratory of Microbial Pathogenesis and Interventions of Fujian Province University, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, 350117, China.

Signal transduction and targeted therapy

|February 15, 2025

概括

Cas12h1是一种CRISPR-Cas酶,作为一个尼克酶,主要切断一个DNA链. 研究人员阐明了它的结构和功能,扩展了用于基因组编辑和诊断的CRISPR工具.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
生物化学生物化学

背景情况:

克里斯普尔-卡斯系统是多样化的,像Cas12h1这样的V型效应器表现出独特的特性.
Cas12h1 识别了一个独特的富含 purin 的原生空间体邻近动图 (PAM).

研究的目的:

为了描述Cas12h1.1.的尼克酶活性.
使用冷EM阐明Cas12h1功能的分子机制.
开发用于增强应用的工程变体.

主要方法:

电子显微镜 (cryo-EM) 用于确定各种功能状态中的结构.
在体外和人体细胞测试中评估尼克酶活性和PAM识别.
理性工程可以创建高保真变体.

主要成果:

Cas12h1 作为一个尼克酶,主要分裂非目标DNA链.
低温EM结构揭示了crRNA成熟,标结合和核酶激活的机制.
Cas12h1 识别了一个广泛的 5 -DHR-3 PAM 序列.
开发了一种高保真性变体,Cas12h1hf,具有改进的不匹配歧视.

结论:

Cas12h1是一种多功能尼克酶,具有独特的PAM偏好和激活机制.
结构洞察力有助于我们更好地了解V型CRISPR-Cas系统.
Cas12h1扩展了基因组编辑和分子诊断的CRISPR工具箱.

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相关概念视频

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...