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

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&#39;s reported high efficiency and specificity in rice14, as well as its ease of use and accessibility to the research group.
<ol>
	<li>Navigate to the NCBI website (https://www.ncbi.nlm.nih.gov/) to retrieve the&#160;OsSBEIIb (LOC4329532) gene in Japonica. Download and access the reference genome within SnapGene software.</li>
	<li>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).</li>
	<li>To validate the exon sequence of the OsSBEIIb gene in X134 by Sanger sequencing, prepare the reaction as follows: 10 &#181;L of polymerase mix buffer with 1 &#181;L of forward primer, 1 &#181;L of Reverse primer, 1 &#181;L of gDNA from X134 tissues and 7 &#181;L of distilled water. Run PCR program as follows: 95 &#176;C, 3 min; (95 &#176;C, 20 s- 56 &#176;C, 30 s- 72 &#176;C, 60 s) with 35 cycles, and 72 &#176;C, 5 min.</li>
	<li>Design the sgRNA on OsSBEIIb exon sequence of X134, adhering to the Tsakirpaloglou et al.14protocol and ensuring the PAM sequence is TTN.</li>
	<li>Modify the sgRNA sequence by adding -ACAC- oligos at the 5&#39; end of the forward primer and adding -GGCC- oligos at the 5&#39; end of the reverse primer. Commercially synthesize the forward/reverse primers (for sgRNA).</li>
	<li>Dissolve the forward and reverse primers to a concentration of 10 &#181;mol/L, take 1 &#181;L of each, and add to 8 &#181;L 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 &#176;C to 16 &#176;C at 0.1 &#176;C/s.</li>
	<li>Assemble the sgRNA into the genome editing vector. Prepare the reaction as follows: 20 &#181;L of mix buffer with 0.5 &#181;L of T4 DNA ligase, 1 &#181;L of Bsa&#160;I, 2 &#181;L of 10x restriction buffer, 2 &#181;L of 10x T4 DNA ligase buffer, 2 &#181;L of annealing primers, 2.5 &#181;L of vector (its volume is adjusted to fit the ratio) and 10 &#181;L 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 &#176;C, 2 min; 16 &#176;C, 3 min) with 30 cycles, and 55 &#176;C, 30 min.</li>
	<li>Transform the resulting vector into E. coli cells as described23.</li>
	<li>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.</li>
</ol>
2. Transformation of Agrobacterium (timing 4 days)
<ol>
	<li>Thaw EHA105 Agrobacterium Competent Cell on ice. Add 1-2 &#181;L of plasmid DNA (containing ~100-200 ng of DNA) to the cell suspension and mix gently.</li>
	<li>Perform heat shock transformation by placing the tube on ice for 5 min, liquid nitrogen for 5 min, heat shock at 37 &#176;C for 5 min, and then in the ice bath for 5 min.</li>
	<li>Add 700 &#181;L of Yeast Extract Peptone (YEP) media, without antibiotic, to the tube and mix gently. Recover the cells in a shaking incubator at 28 &#176;C, 200-250 rpm, for 2-3 h.</li>
	<li>Centrifuge the culture at 2,800 x g for 1 min. Discard most of the supernatant, leaving about 100 &#181;L, 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 &#176;C for 24-48 h.</li>
	<li>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 &#176;C for 3 days.</li>
	<li>Store the transformed Agrobacterium strains as glycerol stocks at -80 &#176;C for further use.</li>
</ol>
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.
<ol>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Extraction of genomic DNA: Follow an established protocol for plant genome DNA extraction26.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Harvesting seeds: Choose the E0 lines exhibiting homozygous frame-shift mutations and plant them in a greenhouse to obtain seeds.</li>
	<li>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).</li>
	<li>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 &#176;C, 3 min; (95 &#176;C, 20 s- 56 &#176;C, 30 s- 72 &#176;C, 60 s) with 35 cycles, and 72 &#176;C, 5 min. This allows for the detection of hygromycin, UBI, and Cas cassette in the plant genome.</li>
	<li>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.</li>
</ol>
5. Resistant starch content measurement (timing 4 days)
<ol>
	<li>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 &#945;-amylose/amyloglucosidase (AMG) procedure outlined below.</li>
	<li>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.</li>
	<li>Transfer the peeled rice seeds into the rice mill and operate it for 60 s to eliminate the aleurone layer, yielding polished rice.</li>
	<li>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.</li>
	<li>Deposit the ground rice powder into a Petri dish and place it in a preheated oven. Set the temperature to 37 &#176;C and allow it to dry for 12 h.</li>
	<li>Precisely weigh 100 mg &#177; 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.</li>
	<li>Add 180 &#181;L of purified water to the tube and boil the samples for 20 min in a water bath.</li>
	<li>Allow the samples to cool down to room temperature, then introduce 4 mL of AMG (3 U/mL) containing pancreatic &#945;-amylase (10 mg/mL) into each tube.</li>
	<li>Securely cap the tubes, mix them on a vortex oscillator, and incubate the tubes at 37 &#176;C for 16 h with continuous agitation.</li>
	<li>Add 4 mL of ethanol and mix using a vortex. Centrifuge the tube at 1,500 x g for 10 min.</li>
	<li>Decant the supernatant, add 2 mL of 50% ethanol followed by 6 mL of 50% IMS, and mix using a vortex and centrifuge.</li>
	<li>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.</li>
	<li>Add 8 mL of 1.2 M sodium acetate buffer (pH 3.8) to each test tube and mix using a magnetic stirrer.</li>
	<li>Immediately introduce 0.1 mL of AMG (3300 U/mL), mix thoroughly, and incubate for 30 min in a water bath at 50 &#176;C with intermittent mixing using a vortex. Then, centrifuge the tube at 1,500 x g for 10 min.</li>
	<li>Transfer 0.1 mL of the supernatant to a glass tube, add 3 mL of Glucose oxidase/peroxidase (GOPOD) reagent and incubate at 50 &#176;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.</li>
	<li>Carefully pipette 200 &#181;L of each blank solution, the solution to be tested, and the standard solution into a 96-well plate.</li>
	<li>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) = &#916;A &#215; F/W &#215;9.27 
	Where &#916;A = absorbance read against the reagent blank; F = conversion from absorbance to micrograms (the absorbance obtained for 100 &#181;g of D-glucose in the GOPOD reaction is determined); W = dry weight of sample analyzed.</li>
</ol>
6. Postprandial blood glucose response (timing 5 days)
<ol>
	<li>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 (&#60; 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.</li>
	<li>Test session procedure (spanning two consecutive days)
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

CRISPR-Based Efficient Genome Editing for Resistant Starch in Rice Breeding

<ol>
	<li>Huang, X., Huang, S., Han, B., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrated+genomics+of+crop+domestication+and+breeding.">The integrated genomics of crop domestication and breeding.</a> Cell. 185 (15), 2828-2839 (2022).</li><li>Labroo, M. R., Studer, A. J., Rutkoski, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosis+and+hybrid+crop+breeding:+A+multidisciplinary+review.">Heterosis and hybrid crop breeding: A multidisciplinary review.</a> Front Genet. 12, 643761 (2021).</li><li>Khalil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+editing+revolution:+review.">The genome editing revolution: review.</a> J Genet Eng Biotechnol. 18 (1), 68 (2020).</li><li>Mojica, F. J., D&#237;ez-Villase&#241;or, C., Garc&#237;a-Mart&#237;nez, J., Soria, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+of+regularly+spaced+prokaryotic+repeats+derive+from+foreign+genetic+elements.">Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.</a> J Mol Evol. 60 (2), 174-182 (2005).</li><li>Rath, D., Amlinger, L., Rath, A., Lundgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR-Cas+immune+system:+biology,+mechanisms+and+applications.">The CRISPR-Cas immune system: biology, mechanisms and applications.</a> Biochimie. 117, 119-128 (2015).</li><li>Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N. J., Nekrasov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editing+plant+genomes+with+CRISPR/Cas9.">Editing plant genomes with CRISPR/Cas9.</a> Curr Opin Biotechnol. 32, 76-84 (2015).</li><li>Koonin, E. V., Makarova, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+Genetic+Elements+and+evolution+of+CRISPR-Cas+systems:+All+the+way+there+and+back.">Mobile Genetic Elements and evolution of CRISPR-Cas systems: All the way there and back.</a> Genome Biol Evol. 9 (10), 2812-2825 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+functions+of+TaGW2+homoeologs+in+wheat+grain+weight+and+protein+content+traits.">Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits.</a> Plant J. 94 (5), 857-866 (2018).</li><li>Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K., Mark Cigan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+maize+directed+by+CRISPR-Cas9+ribonucleoprotein+complexes.">Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes.</a> Nature Comm. 7, 13274 (2016).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9-guide+RNA+directed+genome+editing+in+Soybean.">Cas9-guide RNA directed genome editing in Soybean.</a> Plant Physio. 169 (2), 960-970 (2015).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessment+of+the+four+yield-related+genes+Gn1a,+DEP1,+GS3,+and+IPA1+in+rice+using+a+CRISPR/Cas9+system.">Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system.</a> Front Plant Sci. 7, 377 (2016).</li><li>Karlson, C. K. S., Mohd-Noor, S. N., Nolte, N., Tan, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/dCas9-based+systems:+Mechanisms+and+applications+in+plant+sciences.">CRISPR/dCas9-based systems: Mechanisms and applications in plant sciences.</a> Plants. 10 (10), 2055 (2021).</li><li>Hua, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+the+application+of+genome-editing+technologies+in+crop+breeding.">Perspectives on the application of genome-editing technologies in crop breeding.</a> Mol Plant. 12 (8), 1047-1059 (2019).</li><li>Tsakirpaloglou, N., Septiningsih, E. M., Thomson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+performing+CRISPR/Cas9+genome+editing+for+gene+validation+and+trait+improvement+in+crops.">Guidelines for performing CRISPR/Cas9 genome editing for gene validation and trait improvement in crops.</a> Plants. 12 (20), 3564 (2023).</li><li>Jeon, J. S., Ryoo, N., Hahn, T. R., Walia, H., Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+biosynthesis+in+cereal+endosperm.">Starch biosynthesis in cereal endosperm.</a> Plant Physio Biochem. 48 (6), 383-392 (2010).</li><li>Crofts, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylopectin+biosynthetic+enzymes+from+developing+rice+seed+form+enzymatically+active+protein+complexes.">Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.</a> J Exp Bot. 66 (15), 4469-4482 (2015).</li><li>Butardo, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+down-regulation+of+starch+branching+enzyme+IIb+in+rice+by+artificial+microRNA-+and+hairpin+RNA-mediated+RNA+silencing.">Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing.</a> J Exp Bot. 62 (14), 4927-4941 (2011).</li><li>Baysal, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+rice+starch+branching+enzyme+IIb+triggers+broad+and+unexpected+changes+in+metabolism+by+transcriptional+reprogramming.">Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.</a> Proc Natl Acad Sci USA. 117 (42), 26503-26512 (2020).</li><li>Keenan, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+resistant+starch+in+improving+gut+health,+adiposity,+and+insulin+resistance.">Role of resistant starch in improving gut health, adiposity, and insulin resistance.</a> Adv Nutr. 6 (2), 198-205 (2015).</li><li>Shen, L., Li, J., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistant+starch+formation+in+rice:+Genetic+regulation+and+beyond.">Resistant starch formation in rice: Genetic regulation and beyond.</a> Plant Comm. 3 (3), 100329 (2022).</li><li>Duan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+Cas12i+nuclease+that+is+an+efficient+genome+editing+tool+in+animals+and+plants.">An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants.</a> Innovation (Camb). 5 (2), 100564 (2024).</li><li>Ye, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-BLAST:+A+tool+to+design+target-specific+primers+for+polymerase+chain+reaction.">Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.</a> BMC Bioinform. 13, 134 (2012).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J Vis Exp. (6), e253 (2007).</li><li>Ehrt, S., Schnappinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+plasmids+from+E.+coli+by+alkaline+lysis.">Isolation of plasmids from E. coli by alkaline lysis.</a> Meth Mol Biol. 235, 75-78 (2003).</li><li>Nishimura, A., Aichi, I., Matsuoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+Agrobacterium-mediated+transformation+in+rice.">A protocol for Agrobacterium-mediated transformation in rice.</a> Nat Protoc. 1 (6), 2796-2802 (2006).</li><li>Aboul-Maaty, N. A. F., Oraby, H. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+high-quality+genomic+DNA+from+different+plant+orders+applying+a+modified+CTAB-based+method.">Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method.</a> Bull Natl Res Cent. 43, 25 (2019).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+their+molecular+functions+determining+seed+structure,+components,+and+quality+of+rice.">Genes and their molecular functions determining seed structure, components, and quality of rice.</a> Rice. 15 (1), 18 (2022).</li><li>Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+two+metal-ion+catalysis+of+DNA+cleavage+by+Cas12i2.">Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.</a> Nat Commun. 11 (1), 5241 (2020).</li><li>Lv, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+rice+using+CRISPR/Cas12i3.">Genome editing in rice using CRISPR/Cas12i3.</a> Plant Biotechnol J. 22 (2), 379-385 (2024).</li><li>&#268;erm&#225;k, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multipurpose+toolkit+to+enable+advanced+genome+engineering+in+plants.">A Multipurpose toolkit to enable advanced genome engineering in plants.</a> Plant Cell. 29 (6), 1196-1217 (2017).</li></ol>

The authors have no conflicts of interest to disclose.

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 ...

Traditional breeding relies on introducing traits into crops or producing plant populations with enough variation, which requires long-term field observation1,2. 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 DNA4,5. These DSBs are then repaired by the cell's natural DNA repair mechanisms6,7, 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 content17,18. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 x Taq Plus Master Mix II</td>
			<td>Vazyme Biotech Co.,Ltd</td>
			<td>P213</td>
			<td>&#160;Detecting Single Nucleotide Polymorphism (SNP) of genes</td>
		</tr>
		<tr>
			<td>2,4-Dichlorophenoxyacetic (2,4-D) Acid Solutio</td>
			<td>Phyto Technology</td>
			<td>D309</td>
			<td></td>
		</tr>
		<tr>
			<td>AAM medium</td>
			<td>Shandong Tuopu Biol-engineering Co., Ltd</td>
			<td>M9051C</td>
			<td></td>
		</tr>
		<tr>
			<td>BsaI-HF</td>
			<td>New england biolabs</td>
			<td>R3535</td>
			<td>Bsa I enzyme digestion of the editing vector</td>
		</tr>
		<tr>
			<td>Carbenicillin antibiotics</td>
			<td>Applygen</td>
			<td>APC8250-5</td>
			<td>Selection&#160; medium, regeneration medium</td>
		</tr>
		<tr>
			<td>Casaminoacid</td>
			<td>BBI-Life SciencesCorporation</td>
			<td>A603060-0500</td>
			<td>Callus induction medium, co-cultivation medium, selection medium,regeneration medium</td>
		</tr>
		<tr>
			<td>DH5&#945; Chemically Competent Cell</td>
			<td>Weidi Biotechnology Co., Ltd.</td>
			<td>DL1001</td>
			<td>E. coli competent cells</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>BBI-Life SciencesCorporation</td>
			<td>A610491-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA,disodium salt,dihydrate</td>
			<td>Diamond</td>
			<td>A100105-0500</td>
			<td>CTAB buffer</td>
		</tr>
		<tr>
			<td>EHA105 Chemically Competent Cell</td>
			<td>Weidi Biotechnology Co., Ltd.</td>
			<td>AC1010</td>
			<td>Agrobacterium competent cells</td>
		</tr>
		<tr>
			<td>FastPure Plasmid Mini Kit</td>
			<td>Vazyme Biotech Co.,Ltd</td>
			<td>REC01-100</td>
			<td>Plasmid isolated</td>
		</tr>
		<tr>
			<td>Hygromycin antibiotics</td>
			<td>Yeasen</td>
			<td>60224ES</td>
			<td>co-cultivation medium, selection medium,regeneration medium and root medium</td>
		</tr>
		<tr>
			<td>Kanamycin antibiotics</td>
			<td>Yeasen</td>
			<td>60206ES10</td>
			<td>Selection agrobacterium</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Macklin</td>
			<td>P766798</td>
			<td>CTAB buffer</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Phyto Technology</td>
			<td>G229</td>
			<td>Callus induction medium, co-cultivation medium, selection medium,regeneration medium</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Phyto Technology</td>
			<td>P698</td>
			<td>Callus induction medium, co-cultivation medium, selection medium,regeneration medium</td>
		</tr>
		<tr>
			<td>Mautre dry rice seeds (Xiushui134)</td>
			<td>-</td>
			<td>-</td>
			<td>Japonica varieties for breeding RS rice</td>
		</tr>
		<tr>
			<td>Mill rice mechine</td>
			<td>MARUMASU</td>
			<td>MHR1500A</td>
			<td>To produce white rice</td>
		</tr>
		<tr>
			<td>Murashige Skoog</td>
			<td>Phyto Technology</td>
			<td>M519</td>
			<td>Root medium, regeneration medium</td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Phyto Technology</td>
			<td>I703</td>
			<td>Regeneration medium</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macklin</td>
			<td>S805275</td>
			<td>For&#160; YEP media</td>
		</tr>
		<tr>
			<td>NB Basal Medium</td>
			<td>Phyto Technology</td>
			<td>N492</td>
			<td>Callus induction medium, co-cultivation medium, selection medium,regeneration medium</td>
		</tr>
		<tr>
			<td>Peptone&#160;</td>
			<td>Solarbio</td>
			<td>LA8800</td>
			<td>For&#160; YEP media</td>
		</tr>
		<tr>
			<td>Phytogel</td>
			<td>Shanghai yuanye Bio-Technology Co., Ltd</td>
			<td>S24793</td>
			<td></td>
		</tr>
		<tr>
			<td>Pot&#160;</td>
			<td>Midea group Co.</td>
			<td>MB-5E86</td>
			<td>For cooking rice</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Haier</td>
			<td>BCD-170</td>
			<td>Storage the medium</td>
		</tr>
		<tr>
			<td>Resistant Starch Assay Kit</td>
			<td>Megazyme</td>
			<td>K-RSTAR</td>
			<td>Measurement and analysis resistant starch</td>
		</tr>
		<tr>
			<td>Rifampicin antibiotics</td>
			<td>Sigma</td>
			<td>R3501-250MG</td>
			<td>Selection agrobacterium</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Macklin</td>
			<td>S817439</td>
			<td>For seed sterilization</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Shanghai yuanye Bio-Technology Co., Ltd</td>
			<td>B21647</td>
			<td>Callus induction medium, co-cultivation medium, selection medium,regeneration medium</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New england biolabs</td>
			<td>M0202</td>
			<td>Joining sgRNA to the CEISPRY/Cas-SF01 vector</td>
		</tr>
		<tr>
			<td>The glucose monitor</td>
			<td>Medical Equipment &#38; Supply Co., Ltd</td>
			<td>Xuetang 582</td>
			<td>Detection the blood glucose</td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Macklin</td>
			<td>T766494</td>
			<td>CTAB buffer</td>
		</tr>
		<tr>
			<td>Yeast Agar</td>
			<td>Solarbio</td>
			<td>LA1370</td>
			<td>For&#160; YEP media</td>
		</tr>
		<tr>
			<td>YEP media</td>
			<td>-</td>
			<td>-</td>
			<td>Cultivation of Agrobacterium</td>
		</tr>
	</tbody>
</table>

breeding by design for functional rice with genome editing technologies

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.

Author Spotlight: Streamlining Rice Breeding with CRISPR/Cas for Obtaining Optimal Phenotypic and Agronomic Traits 

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 detailed phenotypic and agronomic trait analysis.We have gone beyond simply demonstrating the feasibility of editing the SBEIIb gene in rice. Our work comprehensively assesses the impact of this editor on the biochemical, molecular, and the agronomic traits of the edited rice. 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 foundational crop varieties that not only have high yield and exceptional quality, but also exhibit strong stress tolerance.

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&#160;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 (<st...

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 ...

breeding-design-for-functional-rice-with-genome-editing

Research

JoVE Journal

Bioengineering

Designing sgRNA and Construction of Vector for Genome Editing to Breed Rice with Resistant Starch

To begin, navigate to the NCBI website to retrieve the required gene from the Japonica Rice variety. Download and access the reference genome within SNAP gene software. Analyze the insertions or deletions and single nucleotide polymorphisms within the exon sequence of O-S-S-B-E 2B from the X134 variety, Comparing it to the reference genome.using the NCBI primer blasts tool, design primers, flanking the exon regions. To validate the exon sequence of the O-S-S-B-E 2B gene in X134 by Sanger sequencing, prepare the reaction and run the PCR program with the appropriate settings. Design the single guide RNA, or S-G-R-N-A on the O-S-S-B-E 2B exon sequence of X134, adhering to the protocol, and ensure that the proto spacer adjacent motif sequence is TTN.Add a CAC 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 S-G-R-N-A 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.

Genotyping Transgenic Plants and Harvesting Seeds for Selecting Rice Varieties Enriched with Resistant Starch

To begin, obtain the SG RNA and transform the Agrobacterium with the plasma 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 SBEIIb 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 frame shift 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 Cascoset 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 SBEIIb mutants and wild type plants in terms of seed setting rate, grains per panicle, and yield per plant.

Resistant Starch Content Measurement in Rice Varieties Developed Through Genome Editing

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 g 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 Hz 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 C for 12 hours. Then add 100 mg of rice powder into a 2 mL microcentrifuge tube, and gently tap the tube.Add 180 L of purified water to the tube and boil the sample in a water bath for 20 minutes. After the sample cools down, introduce 4 mL of AMG containing pancreatic alpha amylase into the tube. Next, mix the contents on a vortex oscillator, and incubate the tube at 37 C for 16 hours with continuous agitation.Now, add 4 mL 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 2 mL of 50%ethanol, followed by 6 mL 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 2 mL of 2 molar potassium hydroxide to it, and stir the sample for 20 minutes to re-suspend flock and dissolve resistant starch. Add 8 mL 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 mL of AMG into the mixture and mix thoroughly. Incubate the sample for 30 minutes at 50 C 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 mL of the supernatant to a fresh glass tube. Add 3 mL of glucose oxidase peroxidase reagent and incubate the sample at 50 C for 20 minutes. Carefully pipette 200 L of each blank sample solution and standard solution into a 96-well plate.Measure the absorbance of each sample at 510 nm against the blank solution and calculate the resistant starch content using the formula. Finally, present the participants with 50 g of prepared rice with 200 mL 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 SBEIIb 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.

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

CRISPR-Based Efficient Genome Editing for Resistant Starch in Rice Breeding

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