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 shift 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.