Development Of A Rice CRISPR/CAS9 System And Functional Studies Of OsPIDa Gene Of Rice

Rice has been domesticated for a long time.It is one of the main staple foods consumed in the world.As human populations growing,maintaining stable grain yield affects living standards and the stability of society.How to improve rice yield has been a major challenge for rice breeders and geneticists.With the completion of the rice genome sequencing and steady progresses in rice functional genomics research,there is a need to develop technologies for rice genetic research and breeding.Finding elite alleles and identifying causing genes for agriculturally important traits have been one of the priorities for rice breeders.Genetic studies and generation of mutant collections have been very effective in cloning important genes.Recently,the development of CRISPR/Cas9 gene-editing technology enables efficient and accurate targeted gene modifications in a variety of organisms including rice.In this research,we report the development of a CRISPR/Cas9 gene editing system that was optimized for editing genes in rice.Furthermore,we greatly expanded the applicability of CRISPR/Cas9 technology in rice so that we can generate various types of mutants in rice.Floret development directly determines the productivity of rice and floret development has been studied extensively in rice.The plant hormone auxin plays an important role in many aspects of plant growth and development,but the molecular mechanisms by which auxin controls rice floret development have remained unsolved.PID gene encodes a protein kinase that regulates auxin signal transduction and auxin transport in Arabidopsis.In this study,we focused on the rice OsPIDa,which is the ortholog of the Arabidopsis PID.We conducted sequence analysis,gene cloning,genetic transformation,and mutant screening to define the genetic and physiological functions of OsPIDa.We analyzed the relationship between OsPIDa and the other homologous genes in the OsPIDa subfamily.We found that OsPIDa is the predominant PID gene in controlling the development of stigma.The main results are listed as the following:1.Established a rice CRISPR/Cas9 gene editing system.Our system has been used to edit many target genes and we observed high efficient gene editing in T0 transgenic rice.The mutation rate was about 60% to 90%.The mutations generated by our gene editing system can be stably transmitted to the next generations.2.Developed different strategies for multiple gene editing in rice.First,we designed a single guide RNA that can target multiple homologous genes,which share the same target sequence.This strategy succeeded in editing multiple genes simultaneously in rice.Secondly,different sgRNA cassettes were produced by different small nuclear RNA promoters for multiple site gene editing.We designed an effective method to construct two sgRNA cassettes to edit two target sequences.Third,we adopted the RGR strategy to assemble different sgRNA cassettes to multiplex gene editing.We were able to use just a single promoter to produce two different RGR cassettes to target two sites.These RGR cassettes also can be combined with other CRISPR/Cas9 constructs to edit even more genes simultaneously.3.Utilized RNA polymerase II promoters,which are the most characterized promoters,to drive sgRNA production in rice.Besides U6 promoter(RNA polymerase III promoter),RGR cassettes also could be driven by Actin1 promoter(RNA polymerase II promoter)to generate high efficient gene editing.4.Developed a technological process for quickly obtaining stable hereditable rice mutant.5.Rice Os PID gene family was identified by sequence alignment and analysis with Arabidopsis PID amino acid sequence in Rice protein sequence database.After Screening the Rice T-DNA and Tos17 insertion lines from Rice Mutant Database(RMD),we found only two mutants AF9 and AF12 with a T-DNA inserted in gene body,respectively,but either the single mutants or double mutants showed no obvious phenotypes.We then screened a rice TILLING mutant library and a series of pid mutants with a single nucleotide change was isolated.Based on amino acid sequence analysis,phenotype analysis,backcross and genetic analysis,two OsPIDa allelic mutations ospida1 and ospida2 had been isolated.Both of the mutants had open hulls and severe defects in flower development.6.Both ospida-1 and ospida-2 only had one amino acid change at different positions.In order to confirm that the observed pid phenotypes were caused by pida,we cloned a 12 Kb genomic fragment that contains the ORF of OsPIDa and transformed it into ospida-1 mutants.After genotyping and genetic analysis T0 and T1 plants,we determined that the phenotype of ospida-1 and ospida-2 were caused by the loss of function of OsPIDa.7.We took advantages of our CRISPR/CAS9 technology and generated new mutant alleles of OsPIDa and its close homolog Os PIDb.The double mutants of ospida and ospidb were also generated by direct transforming the CRISPR/CAS9 construct which target Os PIDb to the callus of ospida-1 or crossing the single CRISPR/CAS9 mutant of Os PIDb to ospida-1.After phenotypic and genetic analysis,we found that the CRISPR alleles of OsPIDa were similar to ospida-1,but with slightly reduced panicle branches.Moreover,we determined that OsPIDa was the key player in control the development of panicle and floret in its subfamily.8.We discovered that OsPIDa is localized in the nucleus,cytoplasm,and plasma membrane.The subcellular localization of OsPIDa was greatly different from the Arabidopsis PID localization,which is exclusively located the the plasma membrane whereas OsPIDa is predominantely localized in the nuclear region.