CRISPR-Cas9 mediated genome editing of () gene in mega rice cultivar MTU1010.

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 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 () gene by using CRISPR-Cas9 gene editing in 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 gene. We selected homozygous 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 . The mutation induced by CRISPR-Cas9 method in gene in rice cv. MTU1010 phenocopied EMS-induced (N69D) mutation reported earlier in japonica cultivar. The 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 is, at least, in part due to downregulation of stomatal developmental genes , and . The Cas9-free mutant exhibited moderate level tolerance to osmotic stress and high level of salt stress in seedling stage. Thus, mutant alleles generated in this study will be useful for improving and grain yield in rice cultivars.