5
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Conclusion
Genome editing technologies based on CRISPR/
Cas9 provide a plethora of applications in human
therapeutics, food science and other fields. To
characterize high-affinity interactions between various
RNA constructs and Cas9, MST offers unprecedented
advantages and delivers precise K
d
s even in the low
picomolar range. Unlike other approaches, K
d
values
can be obtained in less than 10 min.
The high information content of MST data enables
straightforward assay development including
selection of the optimal labeling strategy. For this
interaction system we recommend using a Cy5-
labeled probe, which can be hybridized with an array
of different RNA constructs providing highest flexibility
and most convenient assay set-up.
A B
Figure 4: MST traces (top) and dose-response curves (bottom) for the dgRNA interaction with Cas9. dgRNA2 (A) and dgRNA4
(B) differ significantly in length. The resulting dose-response curves were fitted to a one-site binding model to extract Kd values.
MST experiments were performed with 100 pM Cy5-RNA at LED and 60 % MST power. Fnorm = normalized fluorescence
Figure 5: Dose-response curves for the interaction of various
dgRNAs with Cas9. MST experiments were performed with
100 pM Cy5-RNA at 50 % LED and 60 % MST power. Extracted
K
d
values are summarized in the Table 1.
