Direct observation of the formation of a CRISPR–Cas12a R-loop complex at the single-molecule level

Yang Cui, Yangchao Tang, Meng Liang, Qinghua Ji, Yan Zeng, Hui Chen, Jie Lan, Peng Jin, Lei Wang, Guangtao Song and Jizhong Lou

Here, we develop an optical tweezers-based single-molecule manipulation assay to detect the formation of an R-loop complex in the Cas12a system and characterize its thermodynamic stability. We found that the formation of the R-loop complex induces a two-step unfolding of a DNA hairpin containing the target sequence, the non-target sequence binds loosely to Cas12a and can be easily released from the complex, and the Nuc domain of Cas12a plays key roles in target binding and R-loop formation.

DOI