Chapter 8 - Multiplex genome editing in plants through CRISPR-Cas

Abstract

The CRISPR-Cas9 system is a highly versatile and commonly used tool for genome editing, which finds extensive application in both basic and applied biotechnological research. By modifying the 20 base pair (bp) spacer sequence present in the guide RNA (gRNA), it is possible to reprogram Cas9 to efficiently target distinct DNA sites. The technique of multiplex genome editing enables simultaneous use of multiple gRNAs, thereby enabling the efficient editing of multiple genomic loci. Several methodologies have been devised for in vivo delivery of multiple gRNAs, such as the utilization of multigene cassettes, Csy4-mediated excision, crRNA arrays, ribozyme-flanked gRNAs, tRNA-assisted cleavage of gRNAs, and direct administration of preloaded Cas9 proteins harboring different gRNAs. The technique of multiplex genome editing provides the ability to precisely target multiple DNA sequences, thereby facilitating the elimination of multiple genes or the removal of chromosomal fragments. In order to address the issue of off-target effects, researchers can utilize Cas9-dimers that require the simultaneous expression of two gRNAs. In addition, through the fusion of dCas9 with various functional domains such as activators, repressors, methyltransferases, demethylases, or others, it is possible to achieve accurate regulation of targeted gene expression or DNA methylation patterns. Multiplex genome editing shows great potential in accelerating the functional exploration of plant genes and promoting genetic improvements in agricultural crops.