CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome

Small GTPases are cellular switches that are switched on when bound to GTP and switched off when bound to GDP. Different small GTPase proteins or those with mutations may bind to GTP or GDP with different relative affinities. However, small GTPases generally have very high affinities for guanine nucleotides, rendering it difficult to compare the relative binding affinities for GTP and GDP. Here we developed a method for comparing the relative binding strength of a protein to GTP and GDP using a mant-GDP dissociation assay, whereby the abilities of GTP and GDP to induce the dissociation of bound mant-GDP are compared. This equilibrium type assay is simple, economic, and much faster than obtaining each protein’s affinity for GDP and GTP. The GDP/GTP preference value obtained is useful