1. Single molecule methods for studying CRISPR Cas9-induced DNA unwinding
- Author
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Taekjip Ha, Janice Choi, and Ikenna C Okafor
- Subjects
Nuclease ,biology ,Cas9 ,Helicase ,DNA ,Computational biology ,General Biochemistry, Genetics and Molecular Biology ,Endonuclease ,chemistry.chemical_compound ,Genome editing ,chemistry ,CRISPR-Associated Protein 9 ,biology.protein ,CRISPR ,Guide RNA ,CRISPR-Cas Systems ,DNA Cleavage ,Molecular Biology ,RNA, Guide, Kinetoplastida - Abstract
Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9’s HNH domain before and after cleavage.
- Published
- 2022