CRISPR/Cas9 Tools for Multiplex Genome Editing in Crops

Abstract

AbstractMultiplex gene editing—the genetic modification of multiple sites simultaneously using the CRISPR/Cas9 system—is very useful not only in crop breeding to improve multiple traits but also for functional analysis of gene families. To solve the problem of delivery of many T-DNAs with separated binary vectors into plant cells at the same time, several approaches have been developed to express multiple gRNA cassettes from a single CRISPR/Cas9 vector. Generally, two approaches are used for CRISPR/Cas9 multiplex genome editing in plants: (1) expression of individual gRNA expression cassettes and (2) expression of the multiple gRNAs from one promoter on a single transcript, followed by processing into individual gRNAs. For the latter, several different systems, including endogenous tRNA processing, hammerhead self-cleaving ribozyme, Cys4 endoribonuclease, and intron processing-based systems, have been developed to yield the mature gRNA. Optimization of the Cas9 expression promoter is another important approach to further improve multiplex genome editing, and the tissue-specific expression of Cas9 leads to the induction of precise mutations with low levels of mosaicism. The development of multiplex gene editing provides increased opportunity to produce useful and important crops to enhance productivity and resistance to climate change in the future.KeywordsMultiple genome editingCRISPR/Cas9gRNA cassettesCsy4 ribonuclease