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><tbody><tr><td>2 x Taq Plus Master Mix II</td><td>Vazyme Biotech Co.,Ltd</td><td>P213</td><td>&amp;nbsp;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&amp;nbsp; 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&amp;alpha; Chemically Competent Cell</td><td>Weidi Biotechnology Co., Ltd.</td><td>DL1001</td><td>&lt;em&gt;E. coli &lt;/em&gt;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>&lt;em&gt;Agrobacterium &lt;/em&gt;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&amp;nbsp; 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&amp;nbsp;</td><td>Solarbio</td><td>LA8800</td><td>For&amp;nbsp; 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&amp;nbsp;</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 &amp;amp; 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&amp;nbsp; 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

2.2K Views.  Bellagen Biotechnology Co. Ltd. We werken aan het veredelen van functionele rijst met genoombewerkingstechnologieën. Ons onderzoek gaat niet alleen over het toepassen van een tool voor het bewerken van genen. Het richt zich eerder op de integratie van meerdere aspecten, van genselectie en optimalisatie van bewerkingstools tot gedetailleerde analyse van fenotypische en agronomische eigenschappen.We zijn verder gegaan dan alleen het aantonen van de haalbaarheid van het bewerken van he...

Video: Auteur in de schijnwerpers: Rijstveredeling stroomlijnen met CRISPR/Cas voor het verkrijgen van optimale fenotypische en agronomische eigenschappen 

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Genetics

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Cancer Research

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Biochemistry

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in target genes. TILLING is a highly valuable functional genomics tool for gene validation, especially in small grains in which transformation-based approaches hold serious limitations. Developing a robust mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened to find desired mutations, whereas a high mutagen concentration leads to high mortality in the population, leading to an insufficient number of mutagenized individuals. Once an effective population is developed, there are multiple ways to detect mutations in a gene of interest, and the choice of platform depends upon the experimental scale and availability of resources. The Cel-1 assay and agarose gel-based approach for mutant identification is convenient, reproducible, and a less resource-intensive platform. It is advantageous in that it is simple, requiring no computational knowledge, and it is especially suitable for validation of a small number of genes with basic lab equipment. In the present article, described are the methods for development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay.

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

Developmental Biology

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Biology

Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

Source: Xu, D., et al. <a href="https://www.jove.com/v/61665">Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.</a> J. Vis. Exp. (2020).
In this video, we demonstrate genetic transformation of the rice genome using an Agrobacterium tumefaciens binary vector. This bacterium facilitates the transfer of a foreign DNA plasmid using Agrobacterium virulence proteins.

Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

Source: Xu, D., et al. Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive ...

Breeding by Design for Functional Rice with Genome Editing Technologies

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