Breeding by Design for Functional Rice with Genome Editing Technologies

We are working on breeding functional rice with genome editing technologies. Our research isn't just about applying a gene editing tool, rather, it focuses on the integration of multiple aspects from gene selection and editing tool optimization to detail the phenotypic and agronomic trait analysis. We have gone beyond simply demonstrating the feasibility of editing the SBE2b gene in rice.

Our work will comprehensively assess the impact of this editor on the chemical material and agronomic traits of the edited rice plants. Our lab will focus on several key research questions in the future, aiming to push the limits of genetic editing technologies and crop breeding. Specifically, we are committed to contributing a wide range of functional group varieties that not only have high yield and exceptional quality, but also exhibit a strong stress tolerance.

To begin, navigate to the NCBI website to retrieve the required gene from the Japonica rice variety. Download and access the reference genome within SnapGene software. Analyze the insertions or deletions and single nucleotide polymorphisms within the exon sequence of OSSBE2b from the X134 variety, comparing it to the reference genome.

Using the NCBI Primer-BLAST tool, design primers flanking the exon regions. To validate the exon sequence of the OSSBE2b gene in X134 by Sanger sequencing, prepare the reaction and run the PCR program with the appropriate settings. Design the single guide RNA, or sgRNA, on the OSSBE2b exon sequence of X134, adhering to the Sacher-Paluglu protocol, and ensure that the protospacer adjacent motif sequence is TTN.

Add ACAC oligos at the five prime end of the forward primer and GGCC oligos at the five prime end of the reverse primer. After dissolving the primers, mix one microliter of each primer with eight microliters of anneal buffer. Place the mix in the PCR machine and run the annealing program.

Assemble the sgRNA into the genome editing vector and run the PCR assembly program. Transform the resulting vector into Escherichia coli cells and perform PCR amplification on individual colonies to screen for successful insertions for confirming successful cloning using Sanger sequencing. To begin, obtain the sgRNA and transform the agrobacterium with the plasmid DNA.

Transplant the transgenic plants into pots and grow them for one month in a greenhouse. To assay the mutation type, collect two to three milligrams of fresh leaves from each tiller of a single seedling as a single sample using established protocols. Extract the genomic DNA from the collected leaf samples.

Design PCR primers to amplify the SBE2b gene region surrounding the single guide RNA target site to obtain a 493 base pairs-long amplified fragment. Sequence the PCR fragments directly using the Sanger method to identify mutations. Select the E0 lines exhibiting homozygous frameshift mutations and plant them in a greenhouse to obtain seeds.

Then, harvest leaves from two-week-old seedlings and extract plant genomic DNA. Perform genomic PCR using the appropriate program to detect the presence of hygromycin, UBI, and Cas cassette in the plant genome. Analyze the PCR products using gel electrophoresis to identify lines that show no bands, indicating they are transgene-free E1 lines.

Harvest the seeds from these selected lines. Mutant plants displayed a fully opaque waxy appearance in their grains, unlike the translucent appearance of wild type grains. No significant differences were observed between SBE2B mutants and wild type plants in terms of seed setting rate, grains per panicle, and yield per plant.

To begin, harvest the seeds of mutant and X134 plants and allow them to dry naturally at room temperature until the moisture content reaches approximately 13 to 15%After activating the peeling machine, introduce 10 grams of rice grains into the feeder to efficiently remove the glume shell. Transfer the peeled rice seeds into the rice mill and operate the machine for 60 seconds to eliminate the aleurone layer, yielding polished rice. Next, place the polished rice into the tissue grinder.

Adjust the grinding frequency to 60 hertz and run the grinder for 15 seconds for two cycles to produce rice powder. Deposit the ground rice powder into a Petri dish and place it in a preheated oven set to 37 degrees Celsius for 12 hours. Then, add 100 milligrams of rice powder into a two milliliter microcentrifuge tube and gently tap the tube.

Add 180 microliters of purified water to the tube and boil the sample in a water bath for 20 minutes. After the sample cools down, introduce four milliliters of AMG containing pancreatic alpha amylase into the tube. Next, mix the contents on a vortex oscillator and incubate the tube at 37 degrees Celsius for 16 hours with continuous agitation.

Now, add four milliliters of ethanol and mix the sample using a vortex. Then, centrifuge the tube at 1, 500 G for 10 minutes. After decanting the supernatant, add two milliliters of 50%ethanol, followed by six milliliters of 50%IMS to the tube and mix.

After centrifugating the tube, carefully pour off the supernatant and invert the tube to drain excess liquid. Placing the tube in an ice bath, add two milliliters of two molar potassium hydroxide to it and stir the sample for 20 minutes to resuspend flock and dissolve resistant starch. Add eight milliliters of 1.2 molar sodium acetate buffer, adjusted to pH 3.8 to the tube, and mix using a magnetic stirrer.

Then, immediately introduce 0.1 milliliters of AMG into the mixture and mix thoroughly. Incubate the sample for 30 minutes at 50 degrees Celsius in a water bath with intermittent mixing using a vortex. After incubation, centrifuge the tube at 1, 500 G for 10 minutes.

Now, transfer 0.1 milliliters of the supernatant to a fresh glass tube. Add three milliliters of glucose oxidase/peroxidase reagent and incubate the sample at 50 degrees Celsius for 20 minutes. Carefully pipette 200 microliters of each blank sample solution and standard solution into a 96-well plate.

Measure the absorbance of each sample at 510 nanometers against the blank solution and calculate the resistant starch content using the formula. Finally, present the participants with 50 grams of prepared rice with 200 milliliters of water. After the meal, collect venous blood samples at various time points to perform blood glucose analysis.

Resistant starch content was significantly higher in the SBE2B mutant with 5.2%compared to the wild type. Consumption of the prepared rice led to a slower and reduced glucose response at 15 and 30 minutes, with reductions of 9.7%and 3.7%respectively.