The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes inOryza sativa(rice). In this study, we identifiedMPK1andMPK6(ArabidopsisAtMPK6andAtMPK4orthologs, respectively) as essential genes for rice development by finding the preservation ofMPKfunctional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants ofMPK1were severely dwarfed and sterile, and homozygousmpk1seeds from heterozygous parents were defective in embryo development. By contrast, heterozygousmpk6mutant plants completely failed to produce homozygousmpk6seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely relatedMPKgenes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of riceMPKgenes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality ofMPK1andMPK6in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.Significance Statement
Here we used rice MAP kinase genes to illustrate the utility of a CRISPR/Cas9-based genome editing strategy to genetically characterize and identify essential or lethal genes polycistronic tRNA-gRNA gene editing allows efficient generation of a diversity of allelic mutations at multiple genomic sites and facilitates the functional discovery of redundant and/or essential genes.