Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes
Friday, 2017/07/14 | 07:53:11
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Bastian Minkenberg, Kabin Xie, Yinong Yang PLANT JOURNAL, Volume 89, Issue 3, February 2017 Pages 636–648 SummaryThe 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.
See: http://onlinelibrary.wiley.com/doi/10.1111/tpj.13399/full
Figure 1: The polycistronic tRNA-gRNA gene (PTG) processing system and design of PTGs to target four MPKs. (a) A PTG consists of tRNA-gRNA repeats transcribed by a PolIII promoter. Endogenous RNaseP and RNaseZ recognize the tRNA secondary structure within the primary transcript and cut and release multiple gRNAs. The processed gRNAs complex with Cas9 to target multiple sites. (b) Eight designed gRNAs target four MPK genes. Chromosomal deletion could occur because each gene is targeted by two gRNAs. (c) PTGs co-expressing up to eight gRNAs. PTG2 and PTG6 targeted MPK5. PTG3, PTG4 and PTG5 targeted MPK1, MPK2 and MPK6, respectively. PTG7 and PTG8 targeted MPK1/MPK5 and MPK2/MPK6, respectively, to create double mutants. PTG9 targeted all four MPK genes. |
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