CRISPR/Cas9 Guided Mutagenesis To Improve Rice (Oryza Sativa L.) Grain Yield And Drought Tolerance And Transcriptome And Proteome Profiling Of Induced Mutations

Rice(Oryza sativa L.)is one of the world's most important crops,and breeders are continuously searching for ways to improve grain yield to fulfill the requirements of a constantly rising world population.Conventional breeding methods of crop improvement are not keeping pace with projected increases in population growth.The increase in yield using traditional methods has been below the predicted requirement of 2.4% per year by 2050.Genetic modification mainly for yield improvement and stress tolerance has been transformational,akin to a second green revolution.Drought is the most limiting factor on crop yields globally,although it has not benefited significantly from genetic improvement and continues to be a serious concern for agriculture.Drought-tolerant rice varieties are required to meet the production objective from rainfed areas,and genetic improvement for droughttolerant rice should be a high priority in the future.The completion of rice genome sequencing and steady advancement in functional genomics research facilitated molecular breeding.Finding elite alleles related to important agronomic traits and identifying their functions have been priorities for rice breeders.Genetic studies and the generation of mutant germplasm have been very effective ways to speed up breeding programs.CRISPR/Cas9-mediated genome editing is a potential technique for modifying any genomic site quickly and precisely,and it has become a popular method for molecular breeding in crop plants.Recently,CRISPR/Cas9 gene-editing technology enables efficient and accurate targeted gene modifications in several organisms,including rice.Thus,CRISPR/Cas9 mediated multiplex genome editing creates mutants and provides a valuable resource for functional genomics and rice breeding.Many genes in rice are the negative regulator of rice grain yield.The most direct way of increasing yield and stress tolerance is to knock out or generate mutations in genes that negatively affect yield and stress tolerance.Several genes in the cytochrome P450 family are known to be involved in rice organ growth,but their significance in influencing grain yield is unknown.Drought tolerance is regulated by abscisic acid(ABA),and pyrabactin resistancelike(PYL)proteins are known as ABA receptors.The targeted mutagenesis of yield and stress-related genes is a better way for sustainable yield improvements.This research reports the application of the CRISPR/Cas9 gene-editing system to improve rice grain yield,quality,and drought tolerance.Furthermore,we greatly expanded the applicability of CRISPR/Cas9 technology in rice to study the changes triggered by mutations at the whole transcriptome and proteome levels.In this work,we generated mutants for seven different loci,including OsSPL1,three cytochrome P450 homoeologs(Os03g0603100,Os03g0568400,GL3.2),OsBADH2,Os GS3,and Os PYL9.The genes including OsSPL16/q GW8,Os GS3,and Os PYL9 were targeted independently,whereas,Os03g0603100,Os03g0568400,GL3.2,and OsBADH2 were mutated together.We constructed the binary expression vector to target each gene with at least two single guided RNAs(sg RNAs)and transformed it into a selected rice variety through the Agrobacterium-mediated transformation method.We performed the integrative transcriptome and proteomic analysis for cytochrome P450 homoeologs and OsBADH2 mutants,whereas for OsSPL16,Os GS3,and Os PYL9 mutants,only proteomic analysis was performed.Mutant plants were successfully obtained with two sg RNAs for each gene,and genomic insertions and deletions were characterized.In both target sites of each gene,high mutation efficiency was obtained,and the mutations were mostly deletions.The inheritance of chimeric mutations was unpredictable,whereas homozygous and heterozygous mutations followed Mendelian segregation patterns.Homozygous and heterozygous mutants lacking any T-DNA were screened,and no off-target mutations were detected against each sg RNA.Mutant plants of OsSPL16,Cytochrome P450 homoeologs and OsBADH2,and GS3 showed a significant increase in grain yield with no change in other agronomic parameters.Os PYL9 mutants also showed increased grain yield under drought and well-watered field conditions,whereas decreased flag leaf length and width.Cytochrome P450 homoeologs and OsBADH2 mutants showed increased 2-acetyl-1-pyrroline(2AP)content and grain cell counts.Os PYL9 mutants showed higher ABA,antioxidant activities,chlorophyll content,leaf cuticular wax,and survival rate at the seedling stage,whereas decreased malondialdehyde levels,stomatal conductance,transpiration rate,and vascular bundles.Proteomic analysis of OsSPL16 mutants revealed a total of 44 differentially expressed proteins(DEPs),with 33 and 11 being upregulated and downregulated,respectively.Most of the DEPs were associated with pyruvate dehydrogenase,pyruvate kinase,cell division,and proliferation.KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis revealed that pyruvate metabolism and ubiquitination were all found to be enriched pathways.Gene Ontology(GO)and protein-protein interaction(PPI)network analysis found that most DEPs were related to the pyruvate metabolic process and the pyruvate dehydrogenase complex.RNA-sequencing and proteomic analysis of cytochrome P450 homoeologs and OsBADH2 mutants revealed the differential regulation of genes and proteins associated with the cytochrome P450 family,grain size,and the cell cycle.The genes and proteins associated with the ribosome and photosynthetic pathways were mainly enriched,according to KEGG and hub-gene and protein network analyses.GS3 mutants showed a total of 31 DEPs(20 upregulated and 11 down-regulated)mostly associated with cysteine synthase,cysteine proteinase inhibitor,vacuolar protein sorting-associated,ubiquitin,and DNA ligase.Furthermore,GO analysis found that DEPs were largely enriched in cellular processes,metabolic processes,binding,transmembrane,structural,and catalytic activities.DEPs were found to be primarily engaged in lipid metabolism,particularly oxylipin production,according to KEGG analysis.According to the PPI network,proteins associated with DNA damage-binding,ubiquitin-40 S ribosomal,and cysteine proteinase inhibitor showed a higher degree of interaction.The Os PYL9 mutants showed a total of 324 DEPs(184 upregulated and 140 downregulated),and most of them were relevant to the circadian clock,drought response,and reactive oxygen species.KEGG analysis found that DEPs were only involved in circadian rhythm,and GO analysis revealed that DEPs were enriched in abiotic stimulus-response and abscisic acid-activated signaling pathways.In the PPI network,protein GIGANTEA,adagio-like,and pseudo-response regulator proteins displayed higher interaction.Thus,the results revealed that CRISPR/Cas9-guided mutations could boost rice grain yield,quality,and abiotic stress tolerance.Furthermore,proteomic and transcriptome analyses have a wide range of applications for identifying molecular factors and their dynamic regulation underlying gene mutations.Our findings provide a comprehensive and deep understanding of the role of CRISPR/Cas9 in improving rice yield and quality.This study also provides a convenient and effective way to significantly accelerate the breeding process for the development of high-yielding rice.