Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.
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。
