Efficient CRISPR Gene Editing in Rice Assisted by RUBY

doi:10.13560/j.cnki.biotech.bull.1985.2025-0339

Abstract

Abstract:

Objective To develop a RUBY-assisted CRISPR/Cas9 gene-editing vector (GCR) enabling visual screening of transgenic plants and gene-editing events. Method The GCR vector was constructed by coupling RUBY with the Cas9/gRNA expression cassette. Using OsAGO2, OsAGO3, and OsAGO7 in rice (Oryza sativa) as target genes, two multi-gene editing vectors (GCR-237-1 and GCR-237-2) were designed. These vectors were introduced into rice (Zhonghua 11, ZH11) via Agrobacterium-mediated genetic transformation to generate gene-edited plants. Result After the transformation of GCR-237-1 and GCR-237-2 into rice callus, RUBY markers effectively indicated transgenic positive events, and the target genes of stable transformed red plants were efficiently edited. Conclusion The GCR vector enables effective gene editing in rice, and transgenic or gene-edited plants can be rapidly identified through direct visual observation.

Key words: genetic transformation, gene editing, CRISPR, RUBY, rice (Oryza sativa)

DENG Mei-bi, YAN Lang, ZHAN Zhi-tian, ZHU Min, HE Yu-bing. Efficient CRISPR Gene Editing in Rice Assisted by RUBY[J]. Biotechnology Bulletin, 2025, 41(8): 65-73.

Figures/Tables 10

Table 1 Summary of primers used in this experiment

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Function
DetecOligo1	GTAGGTCTCCTGCAACCAGCAGCGAGGAGGATCCGTTTTAGAGCTAGAAATAGCAAG	构建GCR-237-1载体
DetecOligo2	ACGGGTCTCATCGTGGTCCGTTTGCACCAGCCGGG	构建GCR-237-1载体
DetecOligo3	GTAGGTCTCCACGACGAACTCGGTTTTAGAGCTAGAAATAGCAAG	构建GCR-237-1载体
DetecOligo4	ACGGGTCTCAAAAACAGGGGCTCAATGTGGAAGCATGCACCAGCCGGG	构建GCR-237-1载体
DetecOligo5	GTAGGTCTCCTGCACTTATCTGCGAGACGCAGCTGTTTTAGAGCTAGAAATAGCAAG	构建GCR-237-2载体
DetecOligo6	ACGGGTCTCACTTGCAGATATCTGCACCAGCCGGG	构建GCR-237-2载体
DetecOligo7	GTAGGTCTCCCAAGGAGCTCATGTTTTAGAGCTAGAAATAGCAAG	构建GCR-237-2载体
DetecOligo8	ACGGGTCTCAAAAACAGCTTTAATTTTGGGGGCAATGCACCAGCCGGG	构建GCR-237-2载体
DetecOligo9	CGTGGCGGATTCTCCAAGC	菌检（大肠杆菌和农杆菌）
DetecOligo10	ACCTTGAACTTCTTTGAGGGCAC	菌检（大肠杆菌和农杆菌）
DetecOligo11	CTCAACCCCAAGGCTAACAG	植株转基因阳性鉴定
DetecOligo12	ACCTCAGGGCATCGGAAC	植株转基因阳性鉴定
DetecOligo13	AGCTTGAGATCCTCAACCATGAAT	植株转基因阳性鉴定
DetecOligo14	TTCACACAGGAAACAGCTATGAC	植株转基因阳性鉴定
DetecOligo15	CCACTGCTCTCTTCGATCCC	靶点扩增
DetecOligo16	GGTGCAGGTGGTTGTACTCT	靶点扩增
DetecOligo17	TTTCCCGGTGGAAGAGGAGT	靶点扩增
DetecOligo18	GTGCAAAATCGGCATCCCTC	靶点扩增
DetecOligo19	CGGACAGGTTAAAGCGGTGT	靶点扩增
DetecOligo20	AATGAGCTACAAGGCAAGGG	靶点扩增
DetecOligo21	TGCAGATTCTGGGCAGTACG	靶点扩增
DetecOligo22	TTCTCGAACACGCCGATGAG	靶点扩增
DetecOligo23	AGACCCAGTGCTTCTTGAGC	靶点扩增
DetecOligo24	TGTGGCTGCACAGGTAGAAG	靶点扩增
DetecOligo25	TGCAAGAAGGTATGGTTCTTCAT	靶点扩增
DetecOligo26	GTGCCTCCTCTCCATGACAC	靶点扩增

Table 2 Sequence of target sites

载体编号 Vector number	基因 Gene	靶点序列 Target sequence	限制性内切酶 Restriction enzyme
GCR-237-1	OsAGO2	ACCAGCAGCGAGGAGGATCCAGG	BamH I
	OsAGO3	AACGGACCACGACGAACTCGAGG	Xho I
	OsAGO7	TGCTTCCACATTGAGCCCCTCGG	Ban II
GCR-237-2	OsAGO2	CTTATCTGCGAGACGCAGCTGGG	Pvu II
	OsAGO3	GATATCTGCAAGGAGCTCATCGG	Ban II
	OsAGO7	TTGCCCCCAAAATTAAAGCTTGG	Hind III

Fig. 1 Structural diagram of GCR vectorCas9 (orange box), gRNA (white box), and RUBY (red box)are expressed by the promoter OsUBQ10 P (blue box), and Hsp T is terminator

Fig. 2 Agrobacterium-mediated genetic transformation of riceA: Screening stage. B: Differentiation stage. C: Rooting stage

Fig. 3 Red-phenotype plants obtained after transformation with gene-editing vectors GCR-237-1 and GCR-237-2A: Comparative analysis of wild-type (ZH11, left) and GCR-237-1-transformed edited line A5 (right). B: Comparative analysis of wild-type (ZH11, left) and GCR-237-1-transformed edited line B15 (right). Bar=3 cm

Fig. 4 Positive identification of regenerated plantsM: DNA maker DL2000. WT: Wild type negative control plant (single band). 1-11: Positive regenerated plants (double bands)

Fig. 5 Proportion statistics of Cas9 positive plants and red plantsA: The positive ratio of the Cas9 expression cassette in all plants. The total number of regenerated plants in the three batches of GCR-237-1 and GCR-237-2 are (17, 19 and 20) and (13, 12 and 18), respectively. The number of transgenic positive plants in the three batches of regenerated seedlings of GCR-237-1 and GCR-237-2 are (17, 18, and 18) and (13, 12, and 14), respectively. B: The proportion of red seedlings in Cas9 positive plants. The number of red seedlings among the positive plants in the three batches of GCR-237-1 and GCR-237-2 are (17, 18, and 18) and (13, 12, and 14), respectively. The values in the bar charts are of the mean ± standard error (n=3)

Fig. 6 Identified results of some positive regenerated plants by enzyme digestionA: The mutant of OsAGO3 in GCR-237-1 was identified using Xho I enzyme digestion of PCR products. B: OsAGO7 mutant in GCR-237-2 strain was identified by PCR product digested by Hind III. M: DNA maker DL2000. + sign indicates that the enzyme is cut. In this study, GCR-237-1 strain plants targeted OsAGO2, OsAGO7, and GCR-237-2 strain plantstargeted OsAGO2 and OsAGO3 are directly sent to be sequenced

Fig. 7 Sequencing results of regenerated plants A5 and B15Sequencing results of OsAGO2, OsAGO3, and OsAGO7 in the red plants A5 (A)and B15 (B)

Fig. 8 Editing efficiency statistics of transformed plants and red plantsA: The proportion of edited plants among all plants. The total number of regenerated plants in the three batches of GCR-237-1 and GCR-237-2 are (17, 19, and 20) and (13, 12, and 18), respectively. The number of edited plants in the three batches of regenerated seedlings of GCR-237-1 and GCR-237-2 are (17, 18, and 18) and (13, 12, and 14), respectively. B: The proportion of edited plants among red plants. The number of red seedlings in the three batches of GCR-237-1 and GCR-237-2 are (17, 18, and 18) and (13, 12, and 14), respectively. The number of edited plants among red plants are (17, 18, and 18) and (13, 12, and 14), respectively. The values in the bar charts are of the mean ± standard error (n=3)

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