title: Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes.
abstract: 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 in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, 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, 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 related MPK genes, 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 rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.
authors.affiliation: Intercollege Graduate Degree Program in Plant Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA.|Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, 16802, USA.
authors.forename: Bastian
authors.initials: B
authors.lastname: Minkenberg
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, 16802, USA.
authors.forename: Kabin
authors.initials: K
authors.lastname: Xie
authors.affiliation: Intercollege Graduate Degree Program in Plant Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA.|Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, 16802, USA.
authors.forename: Yinong
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods; D020043:Genes, Essential / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics*; D008957:Models, Genetic; D009154:Mutation; D012275:Oryza / Q000235:genetics* / Q000254:growth & development; D010641:Phenotype; D030821:Plants, Genetically Modified; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
chemical_list: D020928:Mitogen-Activated Protein Kinases
keywords: 
  Oryza sativa
  ; CRISPR/Cas9; essential genes; genome editing; mitogen-activated protein kinase; multi-gene family; technical advance