APPLICATION NOTE
High-Affinity Protein-RNA Interactions
Determination of low-picomolar affinities of sgRNAs and crRNA/tracrRNAs for Cas9
Tatjana Theer#, Jochen Deckert#, Philipp Hadwiger#, Ingo Röhl#, Dennis Breitsprecher*,
Nuska Tschammer*
*NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich
#Axolabs GmbH, Fritz-Hornschuch-Straße 9, 95326 Kulmbach
Abstract
Recent advances in genome engineering technologies based on the RNA-guided
CRISPR endonuclease Cas9 are enabling systematic manipulation of genome
function in a variety of organisms, ranging from bacteria and archaea to humans.
Cas9 is guided to specific locations within a genome by a short RNA search string.
Since genome editing leads to permanent modifications within a genome, the
targeting specificity of Cas9 nucleases is of particular importance, especially for
clinical application and gene editing.
In this work, we demonstrate the versatility of MicroScale Thermophoresis (MST)
to determine the binding affinities of various single-guide RNA (sgRNA), duplex
of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) constructs with
Cas9. The MST assay is superior to classical methods like electrophoretic mobility
shi assay (EMSA) because it allows effortless Kd determination free in solution.
Additionally, MST provides excellent sensitivity while consuming a small amount of
non-hazardous fluorescent-labeled oligos or protein. We analyzed several modified
RNAs, some of which were labeled with the fluorophore Cy5. Using NanoTemper
Technologies Monolith NT.115Pico instrument, we determined the affinities of Cas9
with RNAs differing in length and chemical modification pattern. All interactions
were in the lower picomolar range, the highest measured affinity was 1.0 pM.