Mutant Creatation By Editing Several Endogenous Genes Using Optimized CRISPR System In Wheat

Clustered regularly interspaced short palindromic repeats?CRISPR?is a kind of bacterial defense system that degrades alien invasion DNA and function with various CRISPR-associated proteins?Cas9?.Since its discovery,this system has been widely used in Arabidopsis,rice,and maize for precise gene modification.Because of the complexity of wheat genome,the editing efficiencies of wheat endogenous genes by CRISPR/Cas9 system are not high,and the application of the CRISPR/Cpf1 and CRISPR/xCas9 systems in wheat has not yet been reported.The objectives of this study were to develop a well-performing editing system for wheat and implement it further to edit four important genes of wheat including TaMTL,TaWaxy,TaQ,and TaARG.The main results achieved in this study are as follows:1.CRISPR/SpCas9 expression vectors were constructed firstly by using different promoters?wheat U3 and U6,and rice U6a?to regulate the sgRNA for editing the exogenous GUS gene in a marke-free transgenic line H29 to compare the editing efficiency.The highest editing efficiency of61.4%was obtained in the investigation using TaU3 as promoter,which was the best choice for regulating sgRNA expression.Then,we constructed a CRISPR/SpCas9 vector with two targeting sites by using TaU3 promoter to regulate the sgRNAs within the exogenous GUS gene.The editing efficiency using two sgRNAs was 70.1%,being higher than that using one sgRNA targeting site,and the large fragment deletion efficiency between the two sites was 37.2%.We further constructed CRISPR/LbCpf1 and CRISPR/xCas9 vectors using TaU3 promoter and two targeting sites strategy to editing the exogenous GUS gene and successgully obtained edited plants.The editing efficiencies of the two systems were 3.1%and 1.5%,respectivelly.2.To develop haploid wheat inducer material,the optimized CRISPR/SpCas9 vectors carrying two targeting sites of gM179 and gM471 were applied to edit the TaMTL genes in wheat varieties Fielder and Ningchun4.The editing efficiencies for TaMTL gene in Fielder and Ningchun4 were 57.5%and 55.2%,respectivelly.A large fragement deletion mutation was identified for TaMTL4A and TaMTL4B genes with efficiencies of 10.4%and 8.9%in the edited plants using Ningchun4 as receptor,and it was also identified for TaMTL4A,TaMTL4B and TaMTL4D genes with the efficiencies of 8.9%,5.9%,and 7.9%,respectively,in the edited plants using Fielder as receptor,in which the mutants with reverse insertion of the deleted sequences between the two editing sites within the TaMTL sequences on chromosomes 4A and 4D were identified.Then,the TaMTL-edited plants were identified by phenotype,molecular and cytological methods and the haploid induction rate was found to be 10.0%to 31.6%.The haploid plants showed shorter plant height,fewer tillers,narrow leaves,less chromosomes,and smaller guard cell in length compared with the diploid plants.Additionally,the wheat grains lacking an embryo or endosperm were observed in the TaMTL-edited T₁ generation.3.The optimized CRISPR system was applied to edit the TaAQ and TaDq genes on chromosome 5A and 5D in wheat varieties Fielder and Jimai22.The editing efficiencies of TaQ gene in Fielder and Jimai22 were 45.6%and 44.0%,respectivelly.The TaQ-edited plants displayed a significant difference in spike shape compared with the wild types,and different phenotypes in spike shap were observed in some of single T₀ edited plants.There was not significant change in other characterristics except plant height between the TaDq-edited T₁ plants and the control plants.However,the spike shape and seed setting rate showed a great change in TaAQ-edited plants and TaAQ and TaDq simultaneously edited plants.4.To improve wheat starch quality,the optimized CRISPR system was used to edit the TaWaxy genes in wheat varieties Ningchun4,Fielder,Jimai22,and Zhongmai895.The editing efficiencies of the TaWaxy genes in the four varieties were 80.5%,71.6%,74.7%,and 66.7%,respectivelly.A large fragment deletion mutation was detected in TaWaxy4A,TaWaxy7A,and TaWaxy7D when the two targeting sites gW296 and gW830 were simultaneously edited on any one of chromosomes 4A,7A,or 7D.A mutant with reverse insertion of the deleted sequences within the TaWaxy homologous on chromosomes 4A and 7A was also found in edited plants.The TaWaxy-edited seeds were stained with I₂-KI,it was found that no amylose was existed in the endosperm of the seeds.Transmission electron microscope?TEM?observation showed that the shape and size of starch granules were different between the mutants and controls.5.Promoter TaU6 was used to drive sgRNA in CRISPR/SpCas9 system to edit the TaARG gene in wheat variety Fielder.The editing efficiencies of TaARG2A,TaARG2B,and TaARG2D genes were all less than 10.0%.Chemical analysis of the TaARG homozygous gene mutant revealed that the contents of cadaverine was reduced in the seeds of TaARG-edited plants,while the contents of spermidine and spermine were increased.Additionally,the mino acids and protein contents were reduced in the stems of the mutant plants except for lysine.