Nguyen Giang T, Raju Akshara, Sashital Dipali G
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, United States.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, United States.
Methods Enzymol. 2025;712:117-142. doi: 10.1016/bs.mie.2025.01.034. Epub 2025 Feb 7.
CRISPR-Cas systems use RNA-guided CRISPR-associated (Cas) effectors to neutralize infections in bacteria and archaea. In class 2 CRISPR-Cas systems, Cas9 and Cas12 are single-protein Cas effectors that target double-stranded DNA based on complementarity to the guide RNA before cleaving the target DNA using metal-dependent endonuclease domains. Cas9 and Cas12 proteins can be readily programmed to target any DNA of interest by changing the guiding RNA sequence and have been co-opted for genome editing and other biotechnology purposes. The effect of metal ion concentration is an essential consideration in the physiological role of Cas immunity effectors as well as the biotechnological applications of Cas endonucleases. In this chapter, we describe methods for studying the effect of variable divalent metal ion conditions on the DNA binding and cleavage activities of well-studied Cas9 and Cas12a proteins.
CRISPR-Cas系统利用RNA引导的CRISPR相关（Cas）效应蛋白来抵御细菌和古生菌中的感染。在2类CRISPR-Cas系统中，Cas9和Cas12是单蛋白Cas效应蛋白，它们基于与引导RNA的互补性靶向双链DNA，然后使用金属依赖性核酸内切酶结构域切割靶DNA。通过改变引导RNA序列，Cas9和Cas12蛋白可以很容易地被编程以靶向任何感兴趣的DNA，并已被用于基因组编辑和其他生物技术目的。金属离子浓度的影响是Cas免疫效应蛋白的生理作用以及Cas核酸内切酶生物技术应用中的一个重要考虑因素。在本章中，我们描述了研究可变二价金属离子条件对研究充分的Cas9和Cas12a蛋白的DNA结合和切割活性影响的方法。
