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.