A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

Rapidly growing genetics and bioinformatics studies provide us with an opportunity to obtain a global view of the genetic basis of traits, but also give a challenge to the function validation of candidate genes. CRISPR/Cas9 is an emerging and efficient tool for genome editing. To construct expression clones for the CRISPR/Cas9, most current methods depend on traditional cloning using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on multiple steps of PCR. We developed a system for introducing sgRNA expression cassette(s) directly into plant binary vectors in one step. In this system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR took place. Whilst, two sgRNA expression cassettes were amplified in a single round of PCR. Subsequently, an LR or Golden gate reaction was set up with unpurified PCR product and befitting destination vector. We are able to construct expression clones within 36 h, which greatly improves efficiency and saves cost. Furthermore, the efficiency of this system was verified by an agrobacterium-mediated genetic transformation in rice. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing.