Base Editing Of Maize Acetolactate Synthase Genes To Create Germplasm Resistant To Chlorsulfuron Herbicides

Farmland weeds and crops compete for growth space,light,water,soil,nutrients and other resources lead to grass damage,resulting in crop yield reduction and serious economic losses.With the wide range of weeding spectrum,high activity,good weeding effect and environmentally friendly herbicide has been successfully developed and applied,herbicide use area continues to expand.Herbicides have become the main way of weed control in farmland.However,herbicides cause frequent crop phytotoxicity accidents and become one of the most important problems in agricultural production.Breeding herbicide-resistant crop varieties through conventional and biotechnological methods provides an effective way to solve this problem.Conventional herbicide resistance breeding is limited by difficulties in screening and finding rare mutations.GMO technology requires high time and economic costs environmental and biosafety approval processes and a technology monopoly of multinational companies in the field of patents.Therefore,it is of great industrial application prospect and value to breed resistant varieties of endogenous genes such as mutant maize by genetic editing technology.In this study,a cytosine single-base editing tool was constructed using the maize acetolactate synthase gene（ZmALS1 and ZmALS2）as a target gene,and the 165th proline in maize ZmALS1 and ZmALS2 was replaced by precise single-base editing.Serine,under the condition of not affecting the anabolic activity of its branched amino acids,reduces its sensitivity to sulfonylurea herbicides,and creates a new germplasm of sulfonylurea herbicide-resistant maize.The main results are as follows:1.Rat cytosine deaminase（APOBEC1）was fused to the N-terminus of Cas9（D10A）using the residue peptide XTEN as a linker.A uracil glycosylase inhibitor（UGI）that inhibit base excision repair and a nuclear localization signal（NLS）were fused to the C-terminus of Cas9（D10A）.The APOBEC1-XTEN-Cas9（D10A）-UGI fusion sequence was inserted into the basal vector CUB to construct a cytosine single base editing vector.Cas9（D10A）was initiated with the maize UBI promoter,sgRNA was initiated with the RNA polymerase class III promoter ZmU6-2,and ZmALS1 and ZmALS2were targeted for editing,and finally constructed into an ALS-CBE vector.2.The maize inbred line ZC01 was used as a transforming receptor and transformed by Agrobacterium-mediated stable genetic transformation.38 independent T0 positive transformants were obtained by real-time fluorescence quantitative amplification of the bar gene.T0 positive transformants were selfed or flanking T1 generation.550 T1 transgenic positive plants were obtained by transgenic bar rapid test strip assay.3.Sequencing and verification of target genes of T1 generation transgenic positive plants.The sequencing results showed that there were 13 strains,C₇-G₇,C₇C₈-T₇T₈,and C₇C₈-T₇G₈,each of which had the target C₇-T₇ substitution mutation.These 16 T1 generation lines were derived from 3 lines of the T0 generation.The total mutation rate was 8.0%.The T1 generation positive plants self-crossed to obtain the T2 generation,and the T2 generation plants also detected the above mutations,indicating that the target mutation can be stably inherited to the offspring.4.The effective concentration of chlorsulfuron-methyl herbicide on seedling corn was determined by concentration gradient screening to be 100 mg/L.At this concentration,the herbicide spraying seedling corn plants:the wild type plants sprayed by the herbicides all withered and died,while the mutant plants grew normally and showed tolerance to the chlorsulfuron herbicide.Important biological traits such as 100-grain weight,plant height,ear height and number of leaves were determined for target mutant maize.There were no significant differences in the results of the measurements compared to the wild type plants.5.Online off-target prediction for the designed sgRNA.The top five potential off-target sites were searched and the potential off-target sites were sequenced and verified,and the sequencing results showed no mutations.This is consistent with the latest findings that the CBE system triggers a genome-wide mutation that cannot be predicted by a computer to the off-target site.However,whether the CBE system actually causes off-target and the type and region of off-target mutations in the maize genome is under further investigation.