CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice

Abstract

Rice (Oryza sativa L.) is the predominant food crop for almost half of the world population. In light of the rising world population, it has become imperative to increase rice grain yield per unit area to meet the global food demand. The CRISPR/Cas9 technology has recently emerged as a powerful tool for genetically modifying crops due to its simplicity, precision and genome editing capabilities like knock-in, knock-out, knock-down and expression activation. Recent research revealed that knock-down of rice microRNA, osa-miR1432 can simultaneously enhance resistance towards blast disease and overall yield in rice. Hence the current study, ‘CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice’ was undertaken at the Department of Plant Biotechnology, College of Agriculture, Vellanikkara, Kerala Agricultural University, Thrissur during the period from 2019-2022 with an objective to improve overall yield in rice by enhancing grain filling rate through targeted editing of microRNA osa-miR1432 using CRISPR/Cas9 system. The genomic sequence information of the osa-miR1432 gene was retrieved from rice genome annotation project. The stem-loop sequence of osa-miR1432 available in the microRNA database, miRBase was used for guide RNA (gRNA) designing. The gRNA was designed using the online design tool CRISPR-P v2.0. The best gRNA (osamiR1432 G1) was selected based on its features like location in the genome, on-score value, GC content and no. of off-target sites. Genomic region of osa-miR1432 gene, flanking the gRNA target was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and Clustal Omega analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the gRNA. The development of CRISPR/Cas9 construct was accomplished by annealing of the gRNA strands followed by ligating them into the digested and purified pRGEB32 vector using high fidelity BsaI restriction enzyme. The pRGEB32 vector containing the gRNA construct was cloned into E. coli strain DH5α by transformation using the heat shock method. Plasmid DNA was isolated from the colonies obtained on the transformed plate and were Sanger sequenced using universal M13 reverse primer. The third colony (osamiR1432 G1 #3) was confirmed to be a positive clone after analysing the sequence data. The CRISPR/Cas9 construct was then mobilized into Agrobacterium tumefaciens strain EHA105 using freeze-thaw method. The positive clones were confirmed by plasmid PCR using hygromycin gene specific primers as well as guide sequence specific primers. The positive colony in EHA105 (pRGEB32: osa-miR1432 G1#1) was then used for rice genetic transformation experiments. The rice cultivar Nipponbare (Oryza sativa ssp. japonica cv. Nipponbare) was chosen for the study. Nipponbare seeds were inoculated on to N6 medium supplemented with 2, 4- D (3.0 mg/L) for callus induction. Five days old calli were co-cultivated with Agrobacterium cultures harboring the desired gRNA construct. After 1.5 to 2 days of co-cultivation, the calli were washed thoroughly using bacteriostatic agents augmentin or carbenicillin to remove the excess Agrobacterium load. The calli were then placed on selection medium containing hygromycin and augmentin or carbenicillin. The hygromycin resistant calli showed proliferation after 14 days of incubation. These proliferating microcalli were then transferred to regeneration medium containing NAA (0.02 mg/L) and Kinetin (2.0 mg/L). The regenerated shoots were placed in rooting medium which developed proper roots after one week. The plantlets developed were properly hardened in containment polyhouse. Rice genetic transformation protocol was optimized to suit our laboratory and culture conditions. A total of 25 putative transformant T0 plants were examined for the presence of hygromycin resistance gene and 17 plants were found to be hygromycin positive. PCR amplification of the partial osa-miR1432 gene, flanking the gRNA target region was performed in the hygromycin positive plants and the PCR products were Sanger sequenced to detect mutation. Sequence data analysis using the software Sequencher 5.4.6 detected mutation in seven plants. The detected mutations were single base insertions of A (85.7%) or T (14.3%) exactly at 3 bp upstream to the PAM sequence. The mutation efficiency was found to be 41.18% and the mutations detected were classified as homozygous (71.43%) or heterozygous (28.57%). In the current study, using the CRISPR/Cas9 technology rice plants with mutation in the osa-miR1432 gene were successfully developed which can lead to enhanced grain filling in rice