I 型 A 型 CRISPR-Cas 系统的 RNA 指导的解旋酶-核酸酶蛋白 Cas3 的重建和生化特性分析。
Reconstitution and biochemical characterization of the RNA-guided helicase-nuclease protein Cas3 from type I-A CRISPR-Cas system.
机构信息
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.
出版信息
Methods Enzymol. 2022;673:405-424. doi: 10.1016/bs.mie.2022.03.059. Epub 2022 Apr 27.
Type I is the most prevalent CRISPR system found in nature. It can be further defined into six subtypes, from I-A to I-G. Among them, the Type I-A CRISPR-Cas systems are almost exclusively found in hyperthermophilic archaeal organisms. The system achieves RNA-guided DNA degradation through the concerted action of a CRISPR RNA containing complex Cascade and a helicase-nuclease fusion enzyme Cas3. Here, we summarize assays to characterize the biochemical behavior of Cas3. A steep temperature-dependency was found for the helicase component of Cas3HEL, but not the nuclease component HD. This finding enabled us to establish the correct experimental condition to carry out I-A CRISPR-Cas based genome editing in human cells with extremely high efficiency.
I 型是自然界中最普遍存在的 CRISPR 系统。它可以进一步细分为六个亚型，从 I-A 到 I-G。其中，I-A 型 CRISPR-Cas 系统几乎只存在于嗜热古菌中。该系统通过含有 CRISPR RNA 的复合物 Cascade 和一种解旋酶-核酸酶融合酶 Cas3 的协同作用，实现 RNA 指导的 DNA 降解。在这里，我们总结了用于表征 Cas3 生化行为的实验方法。Cas3HEL 的解旋酶组分表现出陡峭的温度依赖性，但核酸酶组分 HD 则没有。这一发现使我们能够建立正确的实验条件，以极高的效率在人类细胞中进行基于 I-A CRISPR-Cas 的基因组编辑。
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