Multiplex Gene Editing in Rice Using the CRISPR-Cpf1 System.

The class 2/type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been used successfully for simultaneous modification of multiple loci in plants. Two general strategies have been applied to coexpressmultiple single guide RNAs (sgRNAs) to achieve multiplex gene editing in plant cells. One is to construct the multiple guide RNA expression cassettes into separate plasmids when direct gene delivery methods are adopted, such as biolistic bombardment and PEG-mediated protoplast transfection (Shan et al., 2013). The other is to assemble the multiple sgRNAs into a single vector when the Agrobacteria-mediated gene transformation method is used. These multiple sgRNAs can be driven by separate promoters (Ma et al., 2015; Zhang et al., 2016) or expressed as a single transcript for further processing by plant endogenous ribonucleases (Xie et al., 2015). Although these two strategies were reported to be efficient for introducing targeted gene modifications in plant genomes, the construction of CRISPR/ Cas9 vectors was complicated and laborious.