A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity

The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alternative guide RNA validation system called GUIDER. GUIDER features a hairpin loop structure with a proximal guanosine-rich unit, a distal fluorophore unit, and a gRNA-targeting stem component. Cleavage of GUIDER by its complementary RNA-guided Cas9 endonuclease complex yields a fluorescent emission at 525 nm, signaling effective cleavage of the hairpin structure. GUIDER was validated using the model gene target mpcsk9, and it was able to identify the gRNA that could most efficiently cleave the target mpcsk9 gene. The modular design of GUIDER should allow it to have broad applicability in validating gRNAs, and its fluorescent signal output offers a rapid, simple, and quantitative measure of Cas9-mediated DNA cleavage.