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NT-MO-030-01
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MST experiments
The interactions of RNA with the Cas9 were measured
in MST-T buffer, pH 7.8, containing 50 mM Tris-HCl, 150
mM NaCl, 10 mM MgCl2 and 0.05 % Tween-20. The
Cy5-labeled RNAs were each used at a concentration
of 100 pM, Cas9-NT647 was used at 50 pM. The
highest concentration of either unlabeled RNA or
Cas9 used was 10 nM.
Instrumentation and data analysis
Measurements were performed on a NanoTemper
Monolith
TM
NT.115
Pico
instrument. Final Cy5-RNA
concentration of 100 pM yielded fluorescence
intensities of ~ 4000 counts at an LED power of 50
%. The samples were measured at 60 % MST power
with a MST-on time of 15 s and a laser-off time of 1
s. The parameters were deduced in T-Jump and the
resulting dose-response curves fitted to a one-site
binding model to extract K
d
values.
References
1. Walzt, E. Gene-edited CRISPR mushroom escapes US regulation.
Nature News 532, 293 (2016).
2 Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide
RNA and target DNA. Cell 156, 935-949 (2014).
3 Heler, R. et al. Cas9 specifies functional viral targets during CRISPR-
Cas adaptation. Nature 519, 199-202 (2015).
4 Jinek, M. et al. A Programmable Dual-RNA–Guided DNA
Endonuclease in Adaptive Bacterial Immunity. Science 337,
816-821, doi:10.1126/science.1225829 (2012).
5 Jiang, W. & Marraffini, L. A. CRISPR-Cas: New tools for genetic
manipulations from bacterial immunity systems. Annual review of
microbiology 69, 209-228 (2015).
6 Sternberg, S. H. & Doudna, J. A. Expanding the biologist's toolkit
with CRISPR-Cas9. Molecular cell 58, 568-574 (2015).
7 Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications
of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278 (2014).
8 Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. A Cas9–guide
RNA complex preorganized for target DNA recognition. Science 348,
1477-1481 (2015).