10.1021/acschembio.7b00899.s001 Tara R. deBoer Tara R. deBoer Noreen Wauford Noreen Wauford Jing-Yi Chung Jing-Yi Chung Miguel Salvador Torres Perez Miguel Salvador Torres Perez Niren Murthy Niren Murthy A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity American Chemical Society 2018 guanosine-rich unit target mpcsk 9 gene gRNA GUIDER features fluorophore unit genomic target Cas 9-mediated DNA cleavage cleavage assay RNA-guided Cas 9 endonuclease CRISPR-Cas 9 technology hairpin loop structure guide RNA Cleavage-Responsive Stem-Loop Hairpin signal output model gene target mpcsk 9 525 nm genomic engineering Assaying Guide RNA Activity alternative guide RNA validation system hairpin structure endonuclease Cas 9 2018-01-30 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/A_Cleavage-Responsive_Stem-Loop_Hairpin_for_Assaying_Guide_RNA_Activity/5849403 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 <i>in vitro</i> 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 <i>mpcsk9</i>, and it was able to identify the gRNA that could most efficiently cleave the target <i>mpcsk9</i> 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.