Breeding by Design for Functional Rice with Genome Editing Technologies

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

Traditional breeding relies on introducing traits into crops or producing plant populations with enough variation, which requires long-term field observation^1,2. Due to the limitations of traditional breeding, gene editing technology has been developed, which can precisely modify the genome of crops to obtain desired traits of plant populations³. The most widely used gene editing system in plants is CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Cas endonuclease), which relies on a programmable RNA-guided endonuclease to create targeted double-strand breaks (DSBs) in the DNA^4,5. These DSBs are then repaired by the cell's natural DNA repair mechanisms^6,7, often resulting in the introduction of the desired genetic changes. Although this technology has been implemented in various crops, including wheat⁸, maize⁹, soybean¹⁰, and rice¹¹, it is predominantly used to reveal biological problems. Compared to its extensive application in elucidating plant gene functions, research on applying gene editing technologies to crop breeding remains relatively scarce¹².

The process of gene editing in crops typically follows a well-defined workflow that encompasses several key steps¹³. The first step involves identifying the specific gene or genetic region that needs to be modified to achieve the desired trait. Secondly, a gene editing strategy is designed, involving the selection of an appropriate gene editing system (e.g., CRISPR-Cas9 or CRISPR-Cas12) and the design of specific guide RNAs to direct the endonuclease to the target site. Thirdly, the gene editing system is then incorporated into a delivery vector, which is used to introduce the editing machinery into the plant cells. These vectors can be DNA, RNA, or ribonucleoprotein (RNP) complexes. Subsequently, the gene editing vectors are delivered into plant cells using various methods, including Agrobacterium-mediated transformation, particle bombardment, or electroporation. Immediately, the transformed plant cells are cultured under appropriate conditions to generate genetically edited callus or embryogenic tissues. These tissues are then regenerated into whole plants through tissue culture techniques. The regenerated plants are subjected to rigorous molecular characterization to confirm the presence of the desired genetic changes.

A previous article by Tsakirpaloglou et al.¹⁴ provides a broad overview of the gene editing process, from vector design to the generation of edited seedlings, but it does not delve into the detailed analysis of specific traits associated with the targeted gene nor the subsequent evaluation of agronomic performance or functional validation of the edited crops. We have gone beyond simply demonstrating the feasibility of editing a gene in rice. Our work comprehensively assesses the impact of this edit on the biochemical, molecular, and agronomic traits of the edited rice lines. This includes evaluating starch composition, a critical factor influencing grain quality and nutritional value, which has not been extensively explored in previous gene editing studies.

Rice starch typically consists of ~20% amylose and ~80% amylopectin¹⁵. SBEIIb is an enzyme essential for amylopectin synthesis and is expressed in the endosperm¹⁶. The knockdown of OsSBEIIb by hairpin RNA (RNAi) and microRNA expression increased the resistant starch content^17,18. Resistant starch is a substrate starch that cannot be digested and absorbed in the small intestine but is able to be broken down into short-chain fatty acids and gases by certain digestive bacteria in the intestines. Since it cannot be broken down quickly, it has a lower glycemic index compared to other starches and does not cause a rapid rise in blood sugar within a short period of time after eating, which can alleviate diabetes to a certain extent in diet¹⁹. In addition, resistant starch has more physiological functions, such as reducing insulin response, regulating intestinal function, preventing fat accumulation, facilitating weight control, and promoting the absorption of mineral ions. Therefore, it is being widely pursued as a new type of dietary fiber²⁰.

To overcome these challenges and successfully utilize gene editing technologies for breeding functional rice varieties, we have refined and optimized the operational protocols within rice. Our focus has been to meticulously analyze the design of target gene loci, carefully select the most suitable gene editing tools, and conduct rigorous phenotypic analysis throughout the breeding process. As a testament to the power and efficiency of these technologies, we present a case study showcasing the rapid development of a functional rice variety enriched with high-resistant starch. This example underscores the potential of gene editing in accelerating the breeding of functional rice, addressing the current dearth of research in this field.

The study was conducted at Bellagen Biotechnology Co. Ltd in China following the guidelines of the human research ethics committee. Before participating, the study protocol was thoroughly explained to the subjects, who provided informed consent.

1. Designing sgRNA and construction vector (timing 5-7 days)

NOTE: A binary vector was used to express the CRISPR/Cas-SF01 system²¹. Do not have less than 3 nucleotides (nt) mismatch with potential off-target site for sgRNA. The sgRNA adapter needs to complement the sticky end, which is generated by the Bsa I enzyme digestion of the editing vector. The choice of software was based on the software's reported high efficiency and specificity in rice¹⁴, as well as its ease of use and accessibility to the research group.

1. Navigate to the NCBI website (https://www.ncbi.nlm.nih.gov/) to retrieve the OsSBEIIb (LOC4329532) gene in Japonica. Download and access the reference genome within SnapGene software.
2. Analyze the insertions/deletions (InDel) and single nucleotide polymorphisms (SNP) in the exon sequence of OsSBEIIb from the X134 variety, comparing it to the reference genome. Utilize NCBI Primer Blast²² to design primers flanking the exon regions (Supplementary Table 1).
3. To validate the exon sequence of the OsSBEIIb gene in X134 by Sanger sequencing, prepare the reaction as follows: 10 µL of polymerase mix buffer with 1 µL of forward primer, 1 µL of Reverse primer, 1 µL of gDNA from X134 tissues and 7 µL of distilled water. Run PCR program as follows: 95 °C, 3 min; (95 °C, 20 s- 56 °C, 30 s- 72 °C, 60 s) with 35 cycles, and 72 °C, 5 min.
4. Design the sgRNA on OsSBEIIb exon sequence of X134, adhering to the Tsakirpaloglou et al.¹⁴protocol and ensuring the PAM sequence is TTN.
5. Modify the sgRNA sequence by adding -ACAC- oligos at the 5' end of the forward primer and adding -GGCC- oligos at the 5' end of the reverse primer. Commercially synthesize the forward/reverse primers (for sgRNA).
6. Dissolve the forward and reverse primers to a concentration of 10 µmol/L, take 1 µL of each, and add to 8 µL of anneal buffer (Tris-EDTA buffer + 50 mM NaCl) and mix by pipetting. Place in the PCR machine and run the annealing program as follows on a PCR machine: slow cool-down process 95 °C to 16 °C at 0.1 °C/s.
7. Assemble the sgRNA into the genome editing vector. Prepare the reaction as follows: 20 µL of mix buffer with 0.5 µL of T4 DNA ligase, 1 µL of Bsa I, 2 µL of 10x restriction buffer, 2 µL of 10x T4 DNA ligase buffer, 2 µL of annealing primers, 2.5 µL of vector (its volume is adjusted to fit the ratio) and 10 µL of distilled water. In all cases, use a 2:1 insert-to-vector ratio to achieve assembly efficiency. Run the PCR assemble program as (37 °C, 2 min; 16 °C, 3 min) with 30 cycles, and 55 °C, 30 min.
8. Transform the resulting vector into E. coli cells as described²³.
9. Design primers flanking the gRNA insertion site within the plasmid. Using these primers, perform PCR amplification on individual colonies to screen for successful insertions. Perform Sanger sequencing of the PCR products to confirm the successful cloning and isolate plasmid²⁴.

2. Transformation of Agrobacterium (timing 4 days)

1. Thaw EHA105 Agrobacterium Competent Cell on ice. Add 1-2 µL of plasmid DNA (containing ~100-200 ng of DNA) to the cell suspension and mix gently.
2. Perform heat shock transformation by placing the tube on ice for 5 min, liquid nitrogen for 5 min, heat shock at 37 °C for 5 min, and then in the ice bath for 5 min.
3. Add 700 µL of Yeast Extract Peptone (YEP) media, without antibiotic, to the tube and mix gently. Recover the cells in a shaking incubator at 28 °C, 200-250 rpm, for 2-3 h.
4. Centrifuge the culture at 2,800 x g for 1 min. Discard most of the supernatant, leaving about 100 µL, and resuspend the cells. Spread the cells onto YEP agar plates containing 1% antibiotics (Kanamycin and Rifampicin) for plasmid selection. Incubate the plate at 28 °C for 24-48 h.
5. Streak the YEP (with antibiotic resistances) plate with a pipette-tip containing a single colony of Agrobacterium harboring the CRISPR/Cas and transfer the cells into 5 mL of YEP liquid media with appropriate antibiotics. Incubate the liquid at 28 °C for 3 days.
6. Store the transformed Agrobacterium strains as glycerol stocks at -80 °C for further use.

3. Rice transformation by Agrobacterium (timing 3 months)

NOTE: Several plant transformation methods have been reported for the delivery and expression of the foreign DNA sequence in the plant cell²⁵. Considering the single copy integration and low frequency of DSBs in the genome, Agrobacterium-mediated transformation is the method of choice for integrating the expression DNA fragment into rice chromosomes.

1. Perform Agrobacterium-mediated transformation of rice following the protocol in²⁵. The actively growing calli (yellowish white, relatively dry, and 1-3 mm in diameter) is a key point for efficient transformation. Discard the seeds with seedling development or brown callus before infecting the callus with Agrobacterium.

4. Genotyping transgenic plants and seed harvesting (timing 8 months)

NOTE: Two generations of rice will be cultivated to achieve homozygous mutation and foreign DNA-free lines.

1. Sampling seedling tissue: Transplant the transgenic plants into pots and grow them for 1 month in a greenhouse. To assay the mutation type, collect 2-3 mg of fresh leaves from each tiller of a single seedling and combine them into a single sample.
2. Extraction of genomic DNA: Follow an established protocol for plant genome DNA extraction²⁶.
3. Designing the mutation primers (Supplementary Table 1): Design PCR primers to amplify the SBEIIb gene region surrounding the sgRNA target site¹⁴. The amplified fragment is 493 bp in length.
4. Genotyping the mutation: Sequence the PCR fragments directly or clone them into a T-clone vector and sequence using the Sanger method to identify mutations¹⁴.
5. Harvesting seeds: Choose the E0 lines exhibiting homozygous frame-shift mutations and plant them in a greenhouse to obtain seeds.
6. Designing the primers to identify T-DNA: Design three specific primers for the Hygromycin cassette, UBI cassette, and Cas cassette of the construct to identify foreign DNA-free lines among the mutations (Supplementary Table 1).
7. Confirming foreign T-DNA-free lines: Harvest leaves from 2-week-old seedlings and extract plant genome DNA as described in step 4.2. Conduct genomic PCR with the following program: 95 °C, 3 min; (95 °C, 20 s- 56 °C, 30 s- 72 °C, 60 s) with 35 cycles, and 72 °C, 5 min. This allows for the detection of hygromycin, UBI, and Cas cassette in the plant genome.
8. Analyze the PCR products by gel electrophoresis and select lines that show no bands, indicating they are transgene-free E1 lines. Harvest the seeds from these selected lines.

5. Resistant starch content measurement (timing 4 days)

1. Harvest seeds of mutant and X134 plants and allow them to dry naturally at room temperature, maintaining a moisture content of approximately 13%-15%. Determine the resistant starch content using the pancreatic α-amylose/amyloglucosidase (AMG) procedure outlined below.
2. Activate the peeling machine and introduce 10 g of rice grains into the feeder to efficiently remove the glume shell, resulting in processed rice seeds.
3. Transfer the peeled rice seeds into the rice mill and operate it for 60 s to eliminate the aleurone layer, yielding polished rice.
4. Place the polished rice into the tissue grinder, adjusting the grinding frequency to 60 Hz. Run the grinder for 15 s, repeating the cycle 2x to produce rice powder.
5. Deposit the ground rice powder into a Petri dish and place it in a preheated oven. Set the temperature to 37 °C and allow it to dry for 12 h.
6. Precisely weigh 100 mg ± 5 mg of samples and pour directly into a 2.0 mL microcentrifuge tube. Gently tap the tube to ensure that the sample settles at the bottom.
7. Add 180 µL of purified water to the tube and boil the samples for 20 min in a water bath.
8. Allow the samples to cool down to room temperature, then introduce 4 mL of AMG (3 U/mL) containing pancreatic α-amylase (10 mg/mL) into each tube.
9. Securely cap the tubes, mix them on a vortex oscillator, and incubate the tubes at 37 °C for 16 h with continuous agitation.
10. Add 4 mL of ethanol and mix using a vortex. Centrifuge the tube at 1,500 x g for 10 min.
11. Decant the supernatant, add 2 mL of 50% ethanol followed by 6 mL of 50% IMS, and mix using a vortex and centrifuge.
12. Carefully pour off the supernatant and invert the tube to drain any excess liquid. Place the tubes in an ice bath, add 2 mL of 2 M KOH to each tube, and stir the samples for 20 min to resuspend floc and dissolve resistant starch.
13. Add 8 mL of 1.2 M sodium acetate buffer (pH 3.8) to each test tube and mix using a magnetic stirrer.
14. Immediately introduce 0.1 mL of AMG (3300 U/mL), mix thoroughly, and incubate for 30 min in a water bath at 50 °C with intermittent mixing using a vortex. Then, centrifuge the tube at 1,500 x g for 10 min.
15. Transfer 0.1 mL of the supernatant to a glass tube, add 3 mL of Glucose oxidase/peroxidase (GOPOD) reagent and incubate at 50 °C for 20 min. Prepare a reagent blank by mixing 0.1 mL of 100 mM sodium acetate buffer and 3 mL of GOPOD reagent. Prepare standards by mixing 0.1 mL of D-glucose with 3 mL of GOPOD reagent.
16. Carefully pipette 200 µL of each blank solution, the solution to be tested, and the standard solution into a 96-well plate.
17. Measure the absorbance of each sample at 510 nm against the blank solution and calculate the resistant starch content using the provided formula.
    Resistant Starch (g/100 g) = ΔA × F/W ×9.27
    Where ΔA = absorbance read against the reagent blank; F = conversion from absorbance to micrograms (the absorbance obtained for 100 µg of D-glucose in the GOPOD reaction is determined); W = dry weight of sample analyzed.

6. Postprandial blood glucose response (timing 5 days)

1. Use the following inclusion criteria for participants: healthy Asian Chinese adults aged between 18 and 60 years, non-smoker, a body mass index (BMI) between 18.5 and 25 kg/m², and normal blood pressure (< 140/90 mm. Hg). Use the following exclusion criteria: metabolic diseases (such as diabetes, hypertension, etc), gastrointestinal disorders, endocrine system abnormalities, or mental illnesses. A total of 10 participants were screened and recruited.
2. Test session procedure (spanning two consecutive days)
   1. Day 0 (Preparation Day): Ask participants to maintain regular sleep patterns and a normal diet for the first 3 days preceding the test. On the evening prior to the test (after 20:00), ask participants to refrain from high-fiber and high-sugar meals. Vigorous exercise is discouraged on the morning of Day 1.
   2. Day 1 (Testing Day): Prepare white rice by milling the rice seeds, then rinse the white rice 2x. Fill a pot with 1.5 parts water to 1 part white rice. Boil the rice until the water is fully absorbed. Turn off the heat, cover the pot, and let it rest for 10 min.
   3. Randomly assign the 10 participants to two equal groups: one test group to be fed Resistant-Starch white rice and one control group to be fed X134 white rice. Ask the participants to rest for 10 min before the testing begins.
   4. Sterilize fingertips with 75% medical alcohol. Gently press the lancet device against the fingertip and release the spring to prick the skin. Gently squeeze the finger to produce a small blood droplet and touch this droplet to the blood-absorbing end of the test strip in the meter. The meter automatically draws in the blood and starts the testing process. Wait for the blood glucose reading to be displayed.
   5. Rice consumption and blood sampling: Present participants with 50 g of the prepared rice with 200 mL of water and ask them to eat this at a comfortable pace within 5-10 min. After the meal, collect venous blood samples at the following time points: 15 min, 30 min, 60 min, 90 min, and 120 min from the start of the meal. Perform blood glucose analysis as in step 6.2.4.

In the present study, the whole procedures of breeding functional rice were demonstrated by genome editing to obtain stable resistant starch rice varieties. We integrated sgRNA targeting SBEIIb into CRISPR/Cas-SF01 (Supplementary Figure 1), infiltrated rice using Agrobacterium transformation, and obtained E0 generation plants after screening and rooting stages. Plants with loss of gene function were screened, and their resistant starch content was determined after seed harvest (Figure 1). Homozygous mutations (20.8%) were found in the E0 plants by Sanger sequencing, and sbeIIb-1 (-2bp, CC), sbeIIb-2 (-4bp, AGCC), and sbeIIb -3 (-4bp, GTGC) in the target sequence produced frameshift in the coding region giving rise to non-functional proteins (Figure 2, Supplementary Figure 2, and Supplementary Figure 3).

A meticulous selection process was undertaken to identify E1 plants harboring the sbeIIb-1, sbeIIb-2, and sbeIIb-3 mutations in the SBEIIb gene, specifically targeting those lines that were confirmed to be free of T-DNA insertions and exhibiting no evidence of off-target mutations. These plants were then selected for further phenotypic analysis. All plants were grown under natural field conditions and harvested seeds after maturation (Figure 3A). Previous studies indicated that the resistant starch content of rice endosperm determines the degree of transparency exhibited by milled rice²⁷. Grains of sbeIIb mutated plants had a waxy appearance, being white and fully opaque in contrast with the typical translucent appearance of X134 seeds (Figure 3B). The mutated plants were indistinguishable from X134 control in plant height (Supplementary Figure 4A,C), seed-setting rate (Figure 3C), number of grains per panicle (Figure 3D), and yield per plant (Figure 3E), excluding the panicle length decreased 11.9% in the mutants (Supplementary Figure 4B,D).

The RS contents of the sbeIIb is 5.2% compared to the X134 wild-type 0.6% (Figure 4A). To further investigate the functional implications, we evaluated the blood glucose response after consuming the RS rice. Our results indicated that the consumption of RS rice resulted in a 9.7% and 3.7% reduction in glucose response at 15 min and 30 min after rice consumption, respectively (Figure 4B). Importantly, the blood glucose levels of individuals consuming the mutant rice rose more slowly compared to those consuming the X134 rice, suggesting the RS rice's potential applications in glycemic control (Figure 4B).

Figure 1: Model of breeding Resistant Starch rice by genome-editing technology. This model illustrates the key steps: delivery of the vector into the Agrobacterium cell, infection of rice callus by Agrobacterium, regeneration of rice shoots, harvesting of rice grains, and analysis of resistant starch. Please click here to view a larger version of this figure.

Figure 2: Genotype and prediction protein sequence of sbeIIb mutants. Sanger sequencing chromatograms of sbellb-1, sbellb-2, and sbellb-3. Black indicates normal amino acid sequences. Black dotted lines indicate frameshift mutations in amino acids, and red indicates the stop codon. The target position is shown with a red arrow. Please click here to view a larger version of this figure.

Figure 3: Agronomic characteristic and grain quality of sbeIIb mutant. (A) Plant phenotype of sbeIIb. (B) Comparisons of the appearance of X134 and sbeIIb-1 mutants of brown rice. (C-E) Seed setting rate, grains per panicle, and yield per plant of X134 and sbeIIb. The data are the mean ± SDs; NS means no significant difference according to Student's t-test, n=20. Please click here to view a larger version of this figure.

Figure 4: Resistant starch content in mature grains and blood glucose levels. (A) Measurements of resistant starch content in sbeIIb grain, n=3. (B) The blood glucose curve after consuming sbeIIb rice. The data are the mean ± SDs; **, p < 0.01 according to Student's t-test, n=5. Please click here to view a larger version of this figure.

Supplementary Figure 1: Vector of CRISPR/Cas-SF01. Please click here to download this File.

Supplementary Figure 2: The prediction protein sequence of OsSBEIIb and the sbeIIb mutants. The frameshift sequences are marked by red color. Please click here to download this File.

Supplementary Figure 3: Analysis of plant traits of sbeIIb-1 mutant. (A) Growth phenotype of sbeIIb-1 mutant. (B) Spike patterns of sbeIIb-1 mutant. (C) Plant height of sbeIIb-1 mutant. (D) Panicle length of sbeIIb-1 mutant. The data are the means ± SDs; NS means no significant difference; **, p < 0.01 according to Student's t-test. Please click here to download this File.

Supplementary Figure 4. Please click here to download this File.

Supplementary Table 1. List of primers used in this study. Please click here to download this File.

In the process of constructing CRISPR/Cas-SF01-based knockdown vectors, meticulous selection of single-guide RNAs (sgRNA) is pivotal. This necessitates the adoption of sequences that exhibit high editing efficiency with minimal off-target effects. Additionally, the synthesis of targeting primers incorporates short adapter oligos matching splice sites of the vector, ensuring seamless integration. Notably, unlike previous methodologies that required sequential enzymatic digestion, gel purification, and ligation, our study streamlines the process by integrating enzyme cutting and ligation into an all-in-one system. This enhancement improves operational efficiency and reduces the experimental timeline.

When comparing Cas-SF01 to the canonical Cas 9 nuclease, distinct advantages emerge, highlighting its potential in molecular plant breeding. These advantages collectively position Cas-SF01 as a promising tool in agricultural biotechnology. Firstly, Cas-SF01's smaller protein size may enhance cellular delivery efficiency in planta. Secondly, its shorter crRNA sequences facilitate the design of multiplexed targeting strategies, thus enabling more complex genetic modifications²⁸. Thirdly, Cas-SF01 typically induces large-scale mutations compared to Cas9's typical 1 -bp indels, indicating its potential for more substantial genetic alterations²⁹.

In the field of crop gene editing, several factors play a pivotal role in ensuring successful outcomes. Firstly, confirming the gene sequence in edited varieties is imperative to prevent any potential insertions, deletions, or single nucleotide polymorphisms (SNPs) that could hinder sgRNA recognition, thereby affecting the efficiency of the editing process. Secondly, the selection of the appropriate Agrobacterium strain for transformation varies based on the rice cultivar being worked with, highlighting the significance of strain choice for achieving successful gene editing in different rice varieties. Additionally, genotyping of the E0 generation edited seedlings is crucial to guarantee consistency in mutation types. This genotyping should be conducted after 1 month of growth, utilizing DNA extracted from the topmost leaves of each tiller. Owing to the remarkable editing efficiency of Cas-SF01, homozygous edited lines can be secured in the E0 generation, facilitating the selection of T-DNA-free materials in the E1 generation and subsequently expediting the breeding process. Prior to evaluating agronomic traits, it is vital to establish the edited phenotype, as this aids in comprehending its influence on other traits and in determining the desired characteristics of the new cultivar.

There are modifications and troubleshooting of this procedure. When dealing with various crop species, it is indispensable to compare the editing efficiency of Cas9, Cpf1, and Cas-SF01 to ascertain the most appropriate tool for the intended genetic modifications. Furthermore, if homozygous edited lines cannot be identified in the E0 generation, it might be necessary to augment the screening sample size or introduce an extra generation of planting and screening to acquire homozygous T-DNA-free plants.

The selection of the U6 promoter for this study was based on its widespread use and established efficacy in plant genome editing, as evidenced by the recent work by Lv et al.²⁹in efficiently creating functional rice varieties. This promoter has been thoroughly validated and optimized in rice, and we have chosen to follow this well-established path to ensure the reliability and reproducibility of our results. While acknowledging the potential advantages of the CmYLCV promoter, as suggested by Čermák et al.³⁰, we recognize that further investigations comparing the performance of different promoters in our specific experimental system would provide valuable insights.

The use of gene editing technologies to precisely modify functional genomic loci in plants underscores the importance of efficient vector delivery and gene mutation detection. To advance edited plant lines into novel crop cultivars, it is imperative to ascertain the homozygous state of the target gene and select individuals free from exogenous T-DNA fragments. This protocol, developed for a rice variety fortified with high-resistant starch, underscores the potential of gene editing technology in expediting the breeding of functional rice.

The manuscript aims to contribute to the field by presenting a comprehensive and systematic approach to creating functional rice varieties through gene editing. The study goes beyond simply describing the application of a gene editing tool; rather, it focuses on the integration of multiple aspects, from gene selection and editing tool optimization to detailed phenotypic and agronomic trait analysis. In summary, while the work may not introduce entirely new technologies or genes, it contributes to the field by demonstrating a comprehensive and rigorous approach to creating functional rice varieties through gene editing.

The authors have no conflicts of interest to disclose.

This work was supported by funding from the Biological Breeding-Major Projects (2023ZD04074).