Multiplex gene editing provides a powerful tool for targeting members of multigene families. Although previous studies have shown that multiplex gene editing in plants is possible with CRISPR-Cas9, the Cas9 system requires large constructs to express multiple sgRNA cassettes, which are more laborious to construct and could cause unstability and reduce transformation efficiency.
Cpf1 is a dual nuclease that not only cleaves target DNA but also processes its own CRISPR RNA. The study from Prof. ZHU Jiankang’s lab at Shanghai Center for Plant Stress Biology (PSC), Shanghai Institute of Plant Physiology and Ecology (SIPPE), Chinese Academy of Science (CAS), tests FnCpf1 and LbCpf1 for single and multiplex gene editing in rice. The results show that both FnCpf1 and LbCpf1 with their own mature direct repeats induce mutations in transgenic plants. In addition, the LbCpf1 system gives higher editing efficiency in all six tested target sites. Importantly, FnCpf1 and LbCpf1 also show robust activity in multiplex gene editing when expressed together with a single CRISPR array. Moreover, it has been proved that FnCpf1 and LbCpf1 are functional when the direct repeat sequences of their CRISPR arrays are exchanged.
This study demonstrates, for the first time, the feasibility of high efficiency multiplex gene editing in plants using engineered CRISPR-Cpf1 with a simple short DR-guide array. It will significantly simplify multiplex gene editing in plants.
The work, entitled “Multiplex Gene Editing in Rice using the CRISPR-Cpf1 System”, has been published online in Molecular Plant on March 18, 2017.
This work is supported by the Chinese Academy of Sciences.