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

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. An optical tweezers-based single-molecule manipulation assay was developed to detect the formation of an R-loop complex in the CRISPR-Cas12a system.