Experts Develop a Simple and Efficient Cloning System for Genome Editing in Rice

Scientists from Hainan University and Huazhong Agricultural University developed a simple and efficient cloning method for CRISPR-Cas9-mediated genome editing in rice. The results are published in PeerJ Life and Environment.

The most common methods used in constructing closed for CRISPR-Cas9 depend on traditional means such as using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on several steps of polymerase chain reaction. To ease the process, scientists created a system that can be used to introduce sgRNA expression cassettes directly into plant binary vectors in a single step. When using the new system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR takes place. Thus,  two sgRNA expression cassettes were amplified in just one run of PCR. At the same time, an LR or Golden gate reaction was placed with unpurified PCR product and matching destination vector. This leads to the construction of expression cloned in just 36 hours. The efficiency of the system was confirmed through an Agrobacterium-mediated genetic transformation in rice.