Fine-Tuning Rice Amylose Content Via CRISPR/Cas9-Based Genome Editing Technology

Eating and cooking quality（ECQ）is the most important factor for rice grain quality in rice breeding.Amylose content（AC）is a pivotal factor affecting rice ECQ.Rice Waxy（Wx）gene encodes granule-bound starch synthase I（GBSSI）and plays a major role in controlling AC.The application of elite natural Wx alleles has greatly contributed to rice ECQ improvement.Recently,the mining of natural allelic variation of Wx has entered a bottleneck stage,while the drawbacks associated with existing Wx alleles have gradually caught the attention of breeders and researchers.To prevent the high homogeneity of high-ECQ rice breeding and meet the diversified requirements of consumers from different regions,ethnic groups and industry,novel Wx alleles are urgently required by breeders to further improve rice ECQ.The development of CRISPR/Cas technology makes it possible to achieve this goal.In this study,we designed three editing strategies for rice Wx gene,respectively aimed at its 1^st intron,promoter and coding area,so as to harvest a variety of novel Wx alleles for rice ECQ improvement.The main findings are as follows:1.By selecting targets on both sides of the 1st intron of Wx gene,deletion of the 1st intron（S12）of Wx gene was achieved using CRISPR/Cas9 technique in four different backgrounds,X32（indica,Wx^b）,KY131（japonica,Wx^b）,X35（indica,Wx^a）,X55(indica,Wx^lv).In the edited offspring,three large segment deletion mutation types were identified,with 1042bp deletion being the primary mutation type.Analysis of Wx gene expression and grain amylose content showed that after the 1^st intron of Wx^b deleted,Wx gene expression was significantly increased in mutants of both japonica and indica background,had a significant increase in AC,from 12.0%increased to 24.0%;whereas removal of the 1^st intron from Wx^a and Wx^lv alleles did not change Wx gene expression and AC significantly.2.With segmented editing of the Wx gene promoter and the 5’UTR（untranslated region）using CRISPR/Cas9 technology,10 novel Wx alleles S911,S910,S89,S78,S67,S16,S13,S34,S45,S25 with missing large segments at the different region of promoter and the5’UTR of Wx gene were obtained in japonica G17 and indica X32 carrying Wx^b.The results of AAC analysis showed that the AC of S89,S67,S16 and S25 mutants was significantly reduced,with AC similar to that of soft rice.The results of Wx gene expression showed that the changes of AC induced by editing S89,S67,S16 and S25 regions were mainly caused by the decrease of Wx gene expression level.Therefore,editing S89,S67,S16 and S25 regions could effectively regulate the expression of Wx gene and amylose synthesis.3.The editing of S16 section can not only reduce the AC of rice with Wx^b background,but also significantly reduce the AC of rice with Wx^lv background.Futher analysis of S16region showed the core promoter of Wx gene,the transcription start site of Wx gene and the5’G/T splice site of Wx gene 1^st intron were all located within this deleted segment.We speculated that the reasons for the significant reduction of AC caused by editing S16segment may be as follows:Deletion of S16 segment may destroy one or some recognition or binding sites of general transcription factors in RNA polymerase Ⅱ core transcription machinery,thus resulted the lower basic transcription level of Wx,then reduced AC.Deletion of 5’G/T splice site may result in a change in the splicing pattern of the 1^st intron of Wx gene,which may inhibit the splicing of the transcript and lead to a significant decrease in AC.Deletion of the transcription start site of Wx gene may lead to a change in transcription pattern of the Wx gene,resulting in a decrease in AC.4.The S13,S34,S45,and S25 segments are segmented removal of S12 segment(editing the 1^st intron of the Wx gene).Deleton of the entire S12 segment in Wx^b background material caused a significant increase in endosperm AC,while there was no significant change in rice endosperm AC in S13,S34 and S45 regions of S12 region,but deteting S25region would lead to a significant decrease in rice endosperm AC.The reason for such phenotype might be that editing the S25 segment of the 1^st intron of the Wx gene generated a new transcript which would disrupt the normal conformation of the mRNA precursor of the Wx gene,resulting in a decrease in the splicing efficiency of the intron,and ultimately a significant decrease in AC.5.Twenty novel Wx alleles were obtained by single-base editing of six amino acid residues in the middle region of the exon of the Wx gene.There were significant changes in AC in different mutation types compared to wild-type,indicating that base substitution caused mutations in the amino acid residues of GBSSI protein resulted in differences in GBSSI enzyme activity,and consequently different AC.The AC of 20 novel Wx alleles mutants ranged from 2.1%to 28.8%,which greatly enriched the AC variation of rice.The AC of Ex4-80（A/G）,Ex9-173（A/G）,Ex9-174（A/G）and Ex8-20（A/G）mutants is between14.0%and 20.0%,which was in line with the requirements of rice grain AC in the current market.Therefore,mutations at these loci may become excellent resources for improving rice ECQ.