Development of abiotic stress tolerant rice cultivars is necessary for sustainable rice production under the scenario of global climate change, dwindling fresh water resources and increase in salt affected areas. Several genes from rice have been functionally validated by using EMS mutants and transgenics. Often, many of these desirable alleles are not available indica rice which is mainly cultivated, and where available, introgression of these alleles into elite cultivars is a time and labour intensive process, in addition to the potential introgression of non-desirable genes due to linkage. CRISPR-Cas technology helps development of elite cultivars with desirable alleles by precision gene editing. Hence, this study was carried out to create mutant alleles of drought and salt tolerance (DST) gene by using CRISPR-Cas9 gene editing in indica rice cv. MTU1010. We used two different gRNAs to target regions of DST protein that might be involved in protein–protein interaction and successfully generated different mutant alleles of DST gene. We selected homozygous dst mutant with 366 bp deletion between the two gRNAs for phenotypic analysis. This 366 bp deletion led to the deletion of amino acid residues from 184 to 305 in frame, and hence the mutant was named as dst^∆184–305. The dst^∆184–305 mutation induced by CRISPR-Cas9 method in DST gene in indica rice cv. MTU1010 phenocopied EMS-induced dst (N69D) mutation reported earlier in japonica cultivar. The dst^∆184–305 mutant produced leaves with broader width and reduced stomatal density, and thus enhanced leaf water retention under dehydration stress. Our study showed that the reduction in stomatal density in loss of function mutants of dst is, at least, in part due to downregulation of stomatal developmental genes SPCH1, MUTE and ICE1. The Cas9-free dst^∆184–305 mutant exhibited moderate level tolerance to osmotic stress and high level of salt stress in seedling stage. Thus, dst mutant alleles generated in this study will be useful for improving drought and salt tolerance and grain yield in indica rice cultivars.

中文翻译：

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CRISPR-Cas9 介导籼稻巨型水稻品种 MTU1010 耐旱和耐盐 (OsDST) 基因的基因组编辑。

在全球气候变化、淡水资源减少和盐分影响地区增加的情况下，开发耐非生物胁迫的水稻品种对于可持续水稻生产是必要的。水稻的几个基因已通过使用 EMS 突变体和转基因技术进行了功能验证。通常，许多这些理想的等位基因在主要栽培的籼稻中是不可用的，并且在可用的情况下，将这些等位基因渗入到优良品种中是一个时间和劳动力密集的过程，此外由于连锁而可能出现不需要的基因的渗入。 。CRISPR-Cas技术通过精确的基因编辑帮助开发具有理想等位基因的优良品种。因此，本研究旨在利用 CRISPR-Cas9 基因编辑技术在籼稻品种中创建耐旱和耐盐 (DST)基因的突变等位基因。MTU1010。我们使用两种不同的 gRNA 来靶向 DST 蛋白中可能参与蛋白质-蛋白质相互作用的区域，并成功生成了DST基因的不同突变等位基因。我们选择两个 gRNA 之间有 366 bp 缺失的纯合dst突变体进行表型分析。这个 366 bp 的缺失导致框内 184 至 305 个氨基酸残基的缺失，因此该突变体被命名为dst ^{Δ184 – 305}。CRISPR-Cas9方法诱导籼稻DST基因dst ^{Δ184 – 305}突变。早期在粳稻品种中报道了MTU1010 表型复制 EMS 诱导的dst (N69D) 突变。dst ^{Δ184 – 305}突变体产生的叶片宽度更宽，气孔密度降低，从而增强了脱水胁迫下叶片的保水能力。我们的研究表明，dst功能丧失突变体中气孔密度的降低至少部分是由于气孔发育基因SPCH1、MUTE和ICE1的下调所致。不含Cas9的dst ^{Δ184 – 305}突变体在苗期表现出中等水平的渗透胁迫耐受性和高水平的盐胁迫耐受性。因此，本研究中产生的dst突变等位基因将有助于提高籼稻品种的耐旱性和耐盐性以及谷物产量。