Fig. 3: Lyophilization of CRISPR-Cas3 Components for stable storage at room temperature.

A Representative image of lyophilized Cascade proteins targeting EMX1. Proteins were freeze-dried with 10% trehalose and 1× CONAN buffer, then rehydrated with H₂O before the reaction. B Collateral cleavage activity of CRISPR-Cas3 with the ivCascade targeting EMX1 before and after lyophilization. ns: no significance. C Collateral cleavage activity of Cascade stored at room temperature for one week, lyophilized and non-lyophilized. D Collateral cleavage activity of CRISPR-Cas3 with Cas3 protein before and after lyophilization. ns: no significance. E Collateral cleavage activity of CRISPR-Cas3 with lyophilized or non-lyophilized Cas3 stored at 4 °C or room temperature for one week. The RFU ratio was calculated as the signal intensity with lyophilized Cas3 or non-lyophilized Cas3 divided by the signal intensity with Cas3 prepared at the time. F Collateral cleavage activity of lyophilized or non-lyophilized Cas3 and Cascade protein mix stored at 4 °C or room temperature for one week. G Collateral cleavage activity of lyophilized or non-lyophilized Cas3 and Cascade protein mix stored at 4 °C or room temperature for 30 days. The RFU ratio was calculated as the signal intensity with lyophilized Cas3 and Cascade protein mix divided by the signal intensity with Cas3 and Cascade protein mix prepared at the time. In B to G, 100 nM of 60-bp EMX1 oligo DNA was used. In B, C, D, E and F, statistical significances were tested using the two-way ANOVA with Sidak’s post-hoc test. In G, one-way ANOVA followed by Sidak’s multiple comparison test was performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.