The Plant journal : for cell and molecular biology 2016
Discovery of rice essential genes by characterizing CRISPR-edited mutation of closely related rice MAP kinase genes.
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 rice mitogen-activated protein kinase (MPK) genes. In this study, we identified 'term' data-tid='88' href='#term-88'>MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologues, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, 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 two to eight genomic sites in the closely related MPK genes, we demonstrated 45 to 86% frequency of biallelic mutations and successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations and transg'term' data-tid='92' href='#term-92'>ene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals essentiality of MPK1 and MPK6 in rice development and enables functional discovery of the previously inaccessible genes or domains because their phenotypes are masked by lethality or redundancy. This article is protected by copyright. All rights reserved.