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A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity
journal contribution
posted on 2018-01-30, 00:00 authored by Tara R. deBoer, Noreen Wauford, Jing-Yi Chung, Miguel Salvador Torres Perez, Niren MurthyThe
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.
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guanosine-rich unittarget mpcsk 9 genegRNAGUIDER featuresfluorophore unitgenomic targetCas 9-mediated DNA cleavagecleavage assayRNA-guided Cas 9 endonucleaseCRISPR-Cas 9 technologyhairpin loop structureguide RNACleavage-Responsive Stem-Loop Hairpinsignal outputmodel gene target mpcsk 9525 nmgenomic engineeringAssaying Guide RNA Activityalternative guide RNA validation systemhairpin structureendonuclease Cas 9
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