Breeding by Design for Functional Rice with Genome Editing Technologies

Summary

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

Abstract

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.

Introduction

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.

Protocol

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.

Results

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 (

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
2 x Taq Plus Master Mix II	Vazyme Biotech Co.,Ltd	P213	Detecting Single Nucleotide Polymorphism (SNP) of genes
2,4-Dichlorophenoxyacetic (2,4-D) Acid Solutio	Phyto Technology	D309
AAM medium	Shandong Tuopu Biol-engineering Co., Ltd	M9051C
BsaI-HF	New england biolabs	R3535	Bsa I enzyme digestion of the editing vector
Carbenicillin antibiotics	Applygen	APC8250-5	Selection  medium, regeneration medium
Casaminoacid	BBI-Life SciencesCorporation	A603060-0500	Callus induction medium, co-cultivation medium, selection medium,regeneration medium
DH5α Chemically Competent Cell	Weidi Biotechnology Co., Ltd.	DL1001	E. coli competent cells
D-Sorbitol	BBI-Life SciencesCorporation	A610491-0500
EDTA,disodium salt,dihydrate	Diamond	A100105-0500	CTAB buffer
EHA105 Chemically Competent Cell	Weidi Biotechnology Co., Ltd.	AC1010	Agrobacterium competent cells
FastPure Plasmid Mini Kit	Vazyme Biotech Co.,Ltd	REC01-100	Plasmid isolated
Hygromycin antibiotics	Yeasen	60224ES	co-cultivation medium, selection medium,regeneration medium and root medium
Kanamycin antibiotics	Yeasen	60206ES10	Selection agrobacterium
KOH	Macklin	P766798	CTAB buffer
L-Glutamine	Phyto Technology	G229	Callus induction medium, co-cultivation medium, selection medium,regeneration medium
L-Proline	Phyto Technology	P698	Callus induction medium, co-cultivation medium, selection medium,regeneration medium
Mautre dry rice seeds (Xiushui134)	-	-	Japonica varieties for breeding RS rice
Mill rice mechine	MARUMASU	MHR1500A	To produce white rice
Murashige Skoog	Phyto Technology	M519	Root medium, regeneration medium
Myo-inositol	Phyto Technology	I703	Regeneration medium
NaCl	Macklin	S805275	For  YEP media
NB Basal Medium	Phyto Technology	N492	Callus induction medium, co-cultivation medium, selection medium,regeneration medium
Peptone	Solarbio	LA8800	For  YEP media
Phytogel	Shanghai yuanye Bio-Technology Co., Ltd	S24793
Pot	Midea group Co.	MB-5E86	For cooking rice
Refrigerator	Haier	BCD-170	Storage the medium
Resistant Starch Assay Kit	Megazyme	K-RSTAR	Measurement and analysis resistant starch
Rifampicin antibiotics	Sigma	R3501-250MG	Selection agrobacterium
Sodium hypochlorite solution	Macklin	S817439	For seed sterilization
Sucrose	Shanghai yuanye Bio-Technology Co., Ltd	B21647	Callus induction medium, co-cultivation medium, selection medium,regeneration medium
T4 DNA Ligase	New england biolabs	M0202	Joining sgRNA to the CEISPRY/Cas-SF01 vector
The glucose monitor	Medical Equipment & Supply Co., Ltd	Xuetang 582	Detection the blood glucose
Tris-HCL	Macklin	T766494	CTAB buffer
Yeast Agar	Solarbio	LA1370	For  YEP media
YEP media	-	-	Cultivation of Agrobacterium