CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained by using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high yielding rice.

Uncovering the genetic basis for quality traits in the Mediterranean old wheat germplasm and phenotypic and genomic prediction assessment by cross-validation test

“…This dominant protein plays a crucial role in the transportation of storage proteins and is also involved in the hydrolysis of gluten protein responsible for the rheological properties of dough in wheat (Koehler and Ho, 1990;Capocchi et al, 2000;Mikola et al, 2001;Mäkinen and Arendt, 2012). As reported by (Usman et al, 2021), a mutation in the gene encoding Cysteine proteinase inhibitor protein in rice resulted in a positive regulation during grain development and increased grain yield (Usman et al, 2021).…”

Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity

“…The CRISPR/Cas9 technology was then used to create homozygous Gs3 mutation lines with stable inheritance and a long-grain phenotype. Their study offers a simple and effective method for increasing grain production, which may considerably speed up the breeding strategy of long-grain japonica parents and encourage the creation of high-yielding rice [46].…”

CRISPR/Cas9 in plant biotechnology: applications and challenges

“…Other research studies used CRISPR/Cas9 in a similar manner with the aim of producing an observable effect either through gene overexpression or gene knockout in rice. Through these methods, various genes have been identified and successfully manipulated, for example, genes responsible for traits such as pigment (anthocyanin) content (Zheng et al, 2019 ; Hu et al, 2020 ), resistance to disease (bacterial blight and blast disease) (Zhou et al, 2015 ; Wang et al, 2016 ; Kim et al, 2019 ), and grain length (Li et al, 2020a; Usman et al, 2021 ). These findings prove that CRISPR/Cas9 technology is undoubtedly effective in manipulating traits in rice, and it is expected that these methods can be applied to generate more resilient, robust, and nutritious rice, which can drive global sustainability.…”

“…Despite its popularity, there is still a degree of uncertainty when utilizing this method as its success depends on the choice of the plasmid and the cultivar used (Mangena et al, 2017 ). Various studies have reported that the A. rhizogenes -mediated transformation system could have been the cause of low transformation efficiency observed in soybean (Li et al, 2019 ; Bai et al, 2020 ; Zhang et al, 2020a ), rice (Butt et al, 2017 ; Usman et al, 2021 ), and tomato (Ron et al, 2014 ) genome editing. Varying culture conditions can also influence the infection and regeneration rates of the Agrobacterium- infected explants, which affects the reproducibility of the results obtained (Hamada et al, 2018 ) as observed in soybean (Li et al, 2017; Hada, 2018 ; Mangena, 2018 ), clover ( Trifolium subterraneum L.) (Rojo, 2021 ), and cassava (Nyaboga et al, 2015 ).…”