Targeted Deletion of the Wx Gene First Intron via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice With a Wxb Allele, but Not in Rice With a Wxa Allele
Authors: Liu Xingdan , Ding Qi , Wang Wenshu , Pan Yanling , Tan Chao , Qiu Yingbo , Chen Ya , Li Hongjing , Li Yinlong , Ye Naizhong , Xu Nan , Wu Xiao , Ye Rongjian , Liu Jianfeng , Ma Chonglie 
Published: August 2021

Abstract
Background: Rice Waxy (Wx) gene plays a major role in seed amylose synthesis, and consequently controls grain amylose content. The expression of Wx gene is highly regulated at both transcriptional and post-transcriptional levels. Particularly, the GT/TT poplymorphism at the 5` splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often has intermediate to high amylose content, whereas Wxb rice has low to intermediate amylose content. A previous study indicates that rice Wx 1st intron significantly enhances gene expression when it is inserted into the 5` UTR (untranslated region) of a foreign gene. By deleting Wx 1st intron with the CRISPR/Cas9 technology, we intended to create a totally noval Wx allele, and further to investigate how the intron removal affects Wx gene expression and rice grain amylose content.Results: CRISPR/Cas9-mediated targeted deletion of Wx 1st intron was performed on 4 rice inbreds, KY131(Wxb), X32(Wxb), X35(Wxa) and X55(Wxlv). Complete deletion of the 1st intron occurred in 8.6%-11.8% of the primary transformants of these 4 inbreds. Transgene-free, homozygous mutants were obtained. Their grain amylose content and Wx gene expression were analyzed. Compared to the amylose content of wild type plants, mutants’ amylose content was significantly increased from 13.0% to about 24% in KY131 and X32 which both carried the Wxb allele. However, no significant differenece in aylose content was observed between wild type plants and mutants of X35 and X55 which carried the Wxa and Wxlv allele, respectively. Results of Wx gene expression analysis on wild type plants and mutants showed a high consistence with their amylose content results. Mutants of KY131 and X32 accumulated much more steady mRNA transcripts than their wild type plants, while steady mRNA level remained somehow unchanged between wild type plants and mutants of X35 and X55. Grain quality including appearance quality and ECQ(eating and cooking quality) that are tightly linked to amylose content was also evalued on wild type plants and mutants, and data were presented and analyzed. Conclusions:This study presents a novel and fast strategy to increase amylose content for rice inbreds carrying a Wxb allele. Our data strongly suggest that rice Wx 1st intron regulates Wx gene expression mainly at the post-transcriptinal level, not as previously thought that it influences Wx gene transcription as well. In addition, removal of the first intron creates a completely noval Wx allele. Further studies on this new Wx allele would provide invaluable insights into the regulation of Wx gene expression, which will help researchers to engineer more new alleles that leads to the breeding of rice cultivars with better eating and cooking quality.