Figure 5.

NanoDSF assay showed efficient RNP complex formation with equimolar Cas9/dgRNA GFP ratio

(A) Schematic representation of the nanoDSF assay using a constant concentration of Cas9 (0.75 μM) and increased concentrations of dgRNA, expressed as Cas9/dgRNA molar ratios. One Cas9 is only able to load one dgRNA, leading to free molecules for other ratios than equimolar Cas9/dgRNA ratio. The figures were created with https://www.biorender.com/ (Toronto, Canada).

(B) NanoDSF curves and derivative curves used to determine Tm of Cas9 (Tm_Cas9) and RNP complex (Tm_RNP) for each condition tested with dgRNA GFP.

(C) Comparison of RNP complex formation efficiency with increasing concentrations of dgRNA in the same conditions described in A. and B. by analysis of Tm_Cas9 and Tm_RNP with dgRNA GFP. Data are represented as individual replicates with mean ± SEM. No significant difference was noted when nonparametric unpaired Mann-Whitney t-tests (N = 3) were done. dgRNA, dual guide RNA; RNP, ribonucleoprotein complex; Tm, melting temperature; GFP, green fluorescent protein.