Application Notes

Determination of low-picomolar affinities of sgRNAs and crRNA/tracrRNAs for Cas9

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APPLICATION NOTE Monolith is a trademark of NanoTemper Technologies GmbH, Munich, Germany. NanoTemper is a registered trademark of NanoTemper Technologies GmbH, Munich, Germany. ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. NT-MO-030-01 nanotempertech.com 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).

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