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 wi...

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.

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

Breeding by Design for Functional Rice with Genome Editing Technologies

We are working on breeding functional rice with genome editing technologies. Our research isn't just about applying a gene editing tool. Rather, it focuses on the integration of multiple aspects, from gene selection and editing tool optimization to detailed phenotypic and agronomic trait analysis.We have gone beyond simply demonstrating the feasibility of editing the SBEIIb gene in rice. Our work comprehensively assesses the impact of this editor on the biochemical, molecular, and the agronomic traits of the edited rice. Our lab will focus on several key research questions in the future, aiming to push the limits of genetic editing technologies and crop breeding.Specifically, we are committed to contributing a wide range of foundational crop varieties that not only have high yield and exceptional quality, but also exhibit strong stress tolerance.

In the present study, the whole procedures of breeding functional rice were demonstrated by genome editing to obtain stable resistant starch rice varieties. We integrated sgRNA targeting SBEIIb into CRISPR/Cas-SF01 (Supplementary&#160;Figure 1), infiltrated rice using Agrobacterium transformation, and obtained E0 generation plants after screening and rooting stages. Plants with loss of gene function were screened, and their resistant starch content was determined after seed harvest (<st...

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

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

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

Research

JoVE Journal

Bioengineering

338 조회수.  Bellagen Biotechnology Co. Ltd. 게놈 편집 기술을 활용한 기능성 벼 육종에 매진하고 있습니다. 우리의 연구는 단순히 유전자 편집 도구를 적용하는 것에 관한 것이 아닙니다. 오히려, 유전자 선택 및 편집 도구 최적화에서 상세한 표현형 및 농업 경제학적 형질 분석에 이르기까지 여러 측면의 통합에 중점을 둡니다.우리는 단순히 쌀에서 SBEIIb 유전자를 편집하는 것의 타당성?...