Development Of Different Toolkits For CRISPR/Cas9-mediated Rice Genome Editing And Creation Of Novel Wheat Germplasm High In Resistant Starch

Rice and wheat are major staple food crops globaly.In recent years,despite the fact that rice and wheat production have significantly increased over the past decades,they are still facing unprecedented challenges in the context of global climate changes,growing world population,decreased farmland,water shortages as well as environmental and ecological deteriorations.The clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein(Cas)(CRISPR/Cas),as a simple,versatile,robust and cost-effective system for genome manipulation,has recently become the widely used tool for generation of sequence-specific targeted mutagenesis,base editing as well as targeted gene replacement for both functional genomics and crop improvement.Here,we developed different CRISPR/Cas9-mediated genome editing toolkits for targeted mutagenesis,base editing,and gene replacement and created a series of rice and wheat novel germplasm.We also evaluated editing activity of a rice condon optimized x Cas9 variant in rice.The results are as following.1)CRISPR/Cas9-mediated C-T base editing.To test the feasibility and efficiency of base editing capability of n Cas9(D10A)-BE3 in rice,we selected OsSBEIIb and Os PDS as the target genes to generate targeted point mutations.In all three targeted sites,we obtained the desired point mutations,although other types of unexpected mutations were also generated.Finally,we successfully established an efficient base editing system in rice,with the highest efficiency of 20%.We provided a feasible and effective tool for targeted base editing in a crop plant.2)CRISPR/Cas9-mediated targeted gene replacement in rice.Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding.Herein,we established a CRISPR/Cas9-mediated targeted gene replacement system in rice without additional selective pressure by using the Os NRT1.1B gene as an example.We also successfully obtained high nitrogen use efficiency rice plants in just one generation.This work demonstrated the feasibility of replacing any genes with elite alleles within one generation,greatly expanding our ability to improve agriculturally important traits.3)Expanding the scope of CRISPR/Cas9-mediated genome editing in rice.To test the endogenous genome editing capability of x Cas9 in rice,we selected OsSBEIIb and Os PDS as the target genes.Our results showed that x Cas9 enabled editing of target genes containing 5'-NGG-3',5'-NGC-3',5'-NGT-3',5'-NGA-3' and 5'-NNG-3' PAM in rice at a highest editing efficiency of70.8%.Furthermore,to test the endogenous base editing capability of nx Cas9-BE3,we also selected OsSBEIIb as the target genes.Our results demonstrated that the nx Cas9-BE3 could achieve base edting of target genes only containing 5'-NGG-3',and the base editing capability mediated by nx Cas9-BE3 was comparable to n Sp Cas9-BE3.4)The high-amylose wheat generated by CRISPR/Cas9 system.Cereal crops high in amylose content and resistant starch(RS)offer great potential to improve human health and lower the risk of serious noninfectious diseases.Common wheat(Triticum aestivum L.)is a major staple food crop globally.However,the RS contents in the grains of modern wheat varieties are low.Here,we report the generation of high-amylose wheat through targeted mutagenesis of Ta SBEIIa in a modern winter wheat cv Zhengmai 7698(ZM)and a spring wheat cv Bobwhite by CRISPR/Cas9,respectively.We generated a series of transgene-free mutant lines either with partial or triple null Tasbe IIa alleles in ZM and Bobwhite,respectively.Analyses of starch composition,structure and properties revealed that the effects of partial or triple null alleles were dosage dependent with triple null lines demonstrated more profound impacts on starch composition,fine structures of amylopectin,and physiochemical and nutritional properties.The flours of triple null lines possessed significantly increased amylose,RS,protein and soluble pentosan contents which benefit human health.Baking quality analyses indicated that the high-amylose flours may be used as additives or for making cookies.Collectively,we successfully modified the starch composition,structure and properties through targeted mutagenesis of Ta SBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties,and generated transgene-free high-amylose wheat.Our finding provides deep insights on the role of Ta SBEIIa in determining starch composition,structure,properties and end-use quality in different genetic backgrounds,and improving RS content with multiple breeding and end-use applications in cereal crop species through genome editing for health benefits.In conclusion,we established a CRISPR/Cas9 mediated C-T base editing system,and tested the editing window and efficiency.Further,we successfully constructed a CRISPR/Cas9-mediated targeted allele gene replacement system in rice and successfully obtained high nitrogen use efficiency rice plants.Thirdly,we expanded the editing scope of CRISPR/Cas9 in rice by using the x Cas9 variant.Finally,we created high-amylose wheat through targeted mutagenesis of Ta SBEIIa in a modern winter wheat and a spring wheat,respectively,for the first time.Collectively,our researches provided a series of important toolkits and technical support for creating new germplasm quickly and accelerating the molecular breeding process.