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

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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.

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...

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

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

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 ...

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

Research

JoVE Journal

Bioengineering

1.9K 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.

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

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.