RUBY辅助的水稻高效CRISPR基因编辑

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

生物技术通报 ›› 2025, Vol. 41 ›› Issue (8): 65-73.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0339

RUBY辅助的水稻高效CRISPR基因编辑

邓美壁¹(), 严浪²^,³(), 詹志田⁴^,⁵, 朱敏⁶, 和玉兵²^,³()

^1.海南大学热带农林学院，海口 570100
^2.三亚中国农业科学院国家南繁研究院中国农业科学院南繁育种研究中心农业农村部基因编辑创新利用重点实验室（海南），三亚 570024
^3.中国农业科学院作物科学研究所作物基因资源与育种全国重点实验室，北京 100081
^4.南京农业大学作物遗传与种质创新利用全国重点实验室，南京 210095
^5.宁波微萌种业有限公司，宁波 315100
^6.华中农业大学作物遗传改良全国重点实验室，武汉 430070

收稿日期:2025-03-31 出版日期:2025-08-26 发布日期:2025-07-17
通讯作者: 和玉兵，男，博士，副教授，研究方向：基因组编辑技术开发与创新利用；E-mail: heyubing@caas.cn
作者简介:邓美壁，男，硕士研究生，研究方向：基因组编辑技术开发与创新利用；E-mail: 2942331446@qq.com
邓美壁，男，硕士研究生，研究方向：基因组编辑技术开发与创新利用；E-mail: 2942331446@qq.com
基金资助:
国家自然科学基金项目(32200335);三亚中国农业科学院国家南繁研究院南繁专项(YBXM2446);三亚中国农业科学院国家南繁研究院南繁专项(YBXM2505)

Efficient CRISPR Gene Editing in Rice Assisted by RUBY

DENG Mei-bi¹(), YAN Lang²^,³(), ZHAN Zhi-tian⁴^,⁵, ZHU Min⁶, HE Yu-bing²^,³()

^1.College of Tropical Agriculture and Forestry, Hainan University, Haikou 570100
^2.National Nanfan Research Institute (Sanya), Research Center of Breeding in Hainan, Key Laboratory of Gene Editing Technologies (Hainan), Ministry of Agriculture and Rural Affairs, Chinese Academy of Agricultural Sciences (CAAS), Sanya 570024
^3.State Key Laboratory of Crop Gene Resources and Breeding, Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081
^4.State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, Nanjing Agricultural University, Nanjing 210095
^5.Ningbo Weimeng Seed Industry Co. , Ltd. , Ningbo 315100
^6.National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070

Received:2025-03-31 Published:2025-08-26 Online:2025-07-17

摘要/Abstract

摘要：

目的开发一种RUBY辅助的可视化筛选转基因植株并有效指示基因编辑发生的CRISPR/Cas9基因编辑载体GCR。方法将RUBY与Cas9/gRNA的表达盒偶联到一起得到GCR载体，使用水稻的OsAGO2、OsAGO3、OsAGO7为靶基因，分别构建多基因编辑载体GCR-237-1和多基因编辑载体GCR-237-2，以水稻ZH11（Zhonghua 11）为受体材料进行农杆菌介导水稻遗传转化获得基因编辑植株。结果 GCR-237-1和GCR-237-2转化入水稻愈伤后，RUBY标记可以有效指示转基因阳性事件，并且稳定转化得到的红色植株的靶基因被高效编辑。结论利用基因编辑载体GCR可有效编辑水稻基因且通过肉眼直观观察即可有效指示转基因乃至基因编辑植株。

关键词: 遗传转化, 基因编辑, CRISPR, RUBY, 水稻

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)

邓美壁, 严浪, 詹志田, 朱敏, 和玉兵. RUBY辅助的水稻高效CRISPR基因编辑[J]. 生物技术通报, 2025, 41(8): 65-73.

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.

图/表 10

表1 实验所用引物汇总

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

表 2 靶位点序列

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

图1 GCR 载体结构示意图Cas9（橙色盒子）、gRNA（白色盒子）和RUBY（红色盒子）由启动子OsUBQ10 P（蓝色盒子）驱动表达，Hsp T为终止子

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

图2 农杆菌介导的水稻遗传转化过程A：筛选阶段；B：分化阶段；C：生根阶段

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

图3 GCR-237-1和GCR-237-2基因编辑载体转化后得到红色植株A：野生型植株（ZH11，左）和GCR-237-1稳定转化后获得的编辑阳性株系A5 （右）；B：野生型植株（ZH11，左）和GCR-237-2稳定转化后获得的编辑阳性株系B15（右）对比图；标尺3 cm

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

图4 再生植株阳性鉴定M：DNA maker DL2000；WT：野生型阴性对照植株（单条带）；1-11：阳性再生植株（双条带）

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)

图5 Cas9 阳性植株和红色植株比例统计A：所有植株中Cas9表达盒的阳性率。GCR-237-1和GCR-237-2三批次的再生植株总数分别为（17、19、20）和（13、12、18）；GCR-237-1和GCR-237-2三批次再生苗中转基因阳性植株数目分别为（17、18、18）和（13、12、14）。B：Cas9表达盒阳性植株中的红苗比例。GCR-237-1和GCR-237-2三批次阳性植株中的红苗数分别为（17、18、18）和（13、12、14）。柱状图的数值为平均值±标准误（n=3）

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)

图6 部分阳性再生植株的酶切检测结果A：使用Xho I酶切PCR产物，鉴定GCR-237-1株系植株中OsAGO3的突变型；B：使用Hind III酶切PCR产物，鉴定GCR-237-2株系植株中OsAGO7的突变型； M：DNA maker DL2000； “+”表示经过酶切；本研究GCR-237-1株系植株靶向OsAGO2、OsAGO7和GCR-237-2株系植株靶向OsAGO2、OsAGO3直接送测序鉴定

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

图7 再生植株A5和B15的测序结果红色植株A5（A）和B15（B）靶向OsAGO2、OsAGO3和OsAGO7的测序图谱

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)

图 8 转化植株和红色植株编辑效率统计A：所有植株发生编辑植株比例。GCR-237-1和GCR-237-2三批次的再生植株总数分别为（17、19、20）和（13、12、18）；GCR-237-1和GCR-237-2三批次再生苗中发生编辑的植株数目分别为（17、18、18）和（13、12、14）。B：红色植株中发生编辑的植株比例。GCR-237-1和GCR-237-2三批次的红苗数分别为（17、18、18）和（13、12、14），红色植株中发生编辑的植株数目分别为（17、18、18）和（13、12、14）。柱状图的数值为平均值±标准误（n=3）

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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RUBY辅助的水稻高效CRISPR基因编辑

Efficient CRISPR Gene Editing in Rice Assisted by RUBY

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