Breeding by Design for Functional Rice with Genome Editing Technologies

Summary

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

Abstract

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

Introduction

Traditional breeding relies on introducing traits into crops or producing plant populations with enough variation, which requires long-term field observation^1,2. Due to the limitations of traditional breeding, gene editing technology has been developed, which can precisely modify the genome of crops to obtain desired traits of plant populations³. The most widely used gene editing system in plants is CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Cas endonuclease), which relies on a programmable RNA-guided endonuclease to create targeted double-st....

Protocol

The study was conducted at Bellagen Biotechnology Co. Ltd in China following the guidelines of the human research ethics committee. Before participating, the study protocol was thoroughly explained to the subjects, who provided informed consent.

1. Designing sgRNA and construction vector (timing 5-7 days)

NOTE: A binary vector was used to express the CRISPR/Cas-SF01 system²¹. Do not have less than 3 nucleotides (nt) mismatch wi.......

Discussion

In the process of constructing CRISPR/Cas-SF01-based knockdown vectors, meticulous selection of single-guide RNAs (sgRNA) is pivotal. This necessitates the adoption of sequences that exhibit high editing efficiency with minimal off-target effects. Additionally, the synthesis of targeting primers incorporates short adapter oligos matching splice sites of the vector, ensuring seamless integration. Notably, unlike previous methodologies that required sequential enzymatic digestion, gel purification, and ligation, our study .......

Materials

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