Efficient screening of sgRNA mediated by CRISPR/Cas9 via TRV viral vectors in cotton

doi:10.6048/j.issn.1001-4330.2025.06.001

Xinjiang Agricultural Sciences ›› 2025, Vol. 62 ›› Issue (6): 1301-1307.DOI: 10.6048/j.issn.1001-4330.2025.06.001

• Crop Genetics and Breeding·Cultivation Physiology·Physiology and Biochemistry • Previous Articles Next Articles

Efficient screening of sgRNA mediated by CRISPR/Cas9 via TRV viral vectors in cotton

LI Jianping¹(), LIU Zhiqing¹, WANG Weiran¹, WANG Meng¹, ZHOU Zixin¹, YANG Jing¹, ZHU Jiahui¹, LI Yaohua¹, SONG Wu²(), Alifu Aierxi¹, KONG Jie¹()

1. Institute of crops, Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences, Urumqi 830091, China
2. Xinjiang Agricultural Vocational and Technical University, Changji Xinjiang 831100, China

Received:2024-11-11 Online:2025-06-20 Published:2025-07-29
Correspondence author: SONG Wu, KONG Jie
Supported by:
Resource-Sharing Platform Construction Project of Xinjiang Uygur Autonomous Region "Construction of Cotton Biotechnology Breeding Innovation Platform"(PT2313);National Natural Science Foundation of China-Regional Fund Project "Study on the Regulatory Mechanism of COR 27 (GhCOR27) in Response to Cold Stress"(32260449);S & T Innovation Leading Talents Project "Genetic Improvement and Industrialization of Mechanical Cotton Variety"(2022197621);Xinjiang Agricultural Vocational and Technical College(XJNZYKJ202302);Shanghai Cooperation Organization (SCO) Science and Technology Par tnership Project"Introduction and Utilization of High-Quality,Stress-Resistant Crop Germplasm Resources"(2023E01025)

利用TRV病毒载体在棉花中高效筛选CRISPR/Cas9介导的sgRNA

李建平¹(), 刘志清¹, 王为然¹, 王萌¹, 周子馨¹, 杨静¹, 朱家辉¹, 李耀华¹, 宋武²(), 阿里甫·艾尔西¹, 孔杰¹()

1.新疆维吾尔自治区农业科学院棉花研究所,乌鲁木齐 830091
2.新疆农业职业技术大学,新疆昌吉 831100

通讯作者: 宋武,孔杰
作者简介:李建平(1977-),男,新疆乌鲁木齐人,研究员,博士,研究方向为棉花生物技术育种,(E-mail) ljp7786@126.com
基金资助:
新疆维吾尔自治区资源共享平台建设项目“棉花生物技术育种创新平台建设”(PT2313);国家自然科学基金-地区基金项目“棉花冷调控基因COR27(GhCOR27)响应低温胁迫的调控机理研究”(32260449);科技创新领军人才项目“机采棉品种遗传改良与产业化”(2022197621);新疆农业职业技术学院资助课题(XJNZYKJ202302);上海合作组织科技伙伴计划项目“优质、抗逆作物资源材料引进与转化利用”(2023E01025)

Abstract

Abstract:

【Objective】 To screen sgRNAs with high targeted mutagenesis for specific genes in cotton by designing vectors carrying sgRNA expressing cassettes based on TRV virus delivery system to rapidly detect editing efficiency. 【Methods】 The sgRNA cassettes were assembled into the TRV viral vector using the infusion reaction, transiently expressed in Cas9-OE transgenic cotton leaves. Mutageneses for targeted genes were verified by RE-PCR and Sanger sequencing. 【Results】 Different sgRNA delivery systems carrying cotton CLA1 and COR27 gene targeted sgRNA respectively were expressed in Cas9-OE transgenic cotton, and results indicated the frequency of mutations reached more than 65% for all the four systems. All plants showed mutations in the gene with nucleotide deletions being more frequent than insertions or replacements. The editing frequency of delivery system driven by GhU6 promoter was higher (80% or more) than that driven by AtU6 promoter (60%-70%). Moreover, delivery systems with tRNA-gRNA-tRNA (PTG) cassette had slightly higher editing efficiency than those of the other delivery systems. 【Conclusion】 TRV virus-mediated sgRNA delivery systems are functional for gene editing in cotton and act as tools for highly efficient detection of mutations induced by targeted gene sgRNA. The TRV-GhU6:: PTG-sgRNA delivery system has more utility value than others as a tool for high-throughput screening of targeted gene sgRNA.

Key words: TRV; cotton; CRISPR/Cas9; sgRNA; gene editing

摘要：

【目的】设计基于TRV病毒载体介导的基因编辑sgRNA投递系统,快速检测sgRNA基因编辑效率,高效筛选靶标基因sgRNA。【方法】利用infusion反应将靶基因sgRNA组装至TRV病毒表达载体,在Cas9-OE转基因棉花叶片中瞬时表达, 结合PCR和Sanger测序验证sgRNA介导的靶基因突变效果。【结果】不同TRV载体介导的sgRNA投递系统,通过CLA1基因和GhCOR27基因sgRNA验证其编辑效率,突变率均达65%及以上,突变类型多为碱基缺失,少数为碱基插入或替换。棉花GhU6启动子驱动的投递系统编辑效率达80%以上,高于拟南芥AtU6启动子(60%~70%)。同时,组装了tRNA-gRNA-tRNA表达盒(PTG)的投递系统编辑效率略高于其它投递系统。【结论】四套TRV病毒介导的sgRNA投递系统能够在棉花中诱导基因编辑,在筛选验证sgRNA编辑效果方面均能发挥作用,其中TRV-GhU6::PTG-sgRNA投递系统在效率上更具有优势,作为应用于高通量高效筛选靶向基因sgRNA的工具具有更高的潜在应用价值。

关键词: TRV, 棉花, CRISPR/Cas9, sgRNA, 基因编辑

CLC Number:

S188
S562

LI Jianping, LIU Zhiqing, WANG Weiran, WANG Meng, ZHOU Zixin, YANG Jing, ZHU Jiahui, LI Yaohua, SONG Wu, Alifu Aierxi, KONG Jie. Efficient screening of sgRNA mediated by CRISPR/Cas9 via TRV viral vectors in cotton[J]. Xinjiang Agricultural Sciences, 2025, 62(6): 1301-1307.

李建平, 刘志清, 王为然, 王萌, 周子馨, 杨静, 朱家辉, 李耀华, 宋武, 阿里甫·艾尔西, 孔杰. 利用TRV病毒载体在棉花中高效筛选CRISPR/Cas9介导的sgRNA[J]. 新疆农业科学, 2025, 62(6): 1301-1307.

Figures/Tables 7

References 23

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引物名称 Primers name	引物序列 Primer sequence(5'-3')	注释 Annotation
AtU6-CLA1-F	tcccttcggaattctctagaTATGCTCGCGGAATGATCAG	正向引物小写字母为AtU6载体序列,大写字母为CLA1 sgRNA序列。反向引物小写字母为TRV2载体序列,大写字母为CLA1 sgRNA序列
CLA1-R	CTGATCATTCCGCGAGCATAggaggccttctagagaattc
GhU6-CLA1-F	aagcgaaagaagcatTATGCTCGCGGAATGATCAG	正向引物小写字母为GhU6载体序列,大写字母为CLA1 sgRNA序列,反向引物为CLA1-R
PGT-CLA1-F	caaagcaccagtggtctagtTATGCTCGCGGAATGATCAG	正向引物小写字母为tRNA序列,大写字母为CLA1 sgRNA序列,反向引物为CLA1-R
AtU6-COR27-F	tcccttcggaattctctagaTGAGTTGACTCAAACCAACT	注释同AtU6-CLA1-F,R。靶标基因为GhCOR27
COR27-R	AGTTGGTTTGAGTCAACTCAggaggccttctagagaattc	注释同CLA1-R,靶标基因为GhCOR27
GhU6-COR27-F	aagcgaaagaagcatTGAGTTGACTCAAACCAACT	注释同GhU6-CLA1-F,R,靶标基因为GhCOR27
PGT-COR27-F	caaagcaccagtggtctagtTGAGTTGACTCAAACCAACT	注释同PGTR-CLA1-F,靶标基因为GhCOR27

引物名称 Primers name	引物序列 Primer sequence(5'-3')	注释 Annotation
AtU6-CLA1-F	tcccttcggaattctctagaTATGCTCGCGGAATGATCAG	正向引物小写字母为AtU6载体序列,大写字母为CLA1 sgRNA序列。反向引物小写字母为TRV2载体序列,大写字母为CLA1 sgRNA序列
CLA1-R	CTGATCATTCCGCGAGCATAggaggccttctagagaattc
GhU6-CLA1-F	aagcgaaagaagcatTATGCTCGCGGAATGATCAG	正向引物小写字母为GhU6载体序列,大写字母为CLA1 sgRNA序列,反向引物为CLA1-R
PGT-CLA1-F	caaagcaccagtggtctagtTATGCTCGCGGAATGATCAG	正向引物小写字母为tRNA序列,大写字母为CLA1 sgRNA序列,反向引物为CLA1-R
AtU6-COR27-F	tcccttcggaattctctagaTGAGTTGACTCAAACCAACT	注释同AtU6-CLA1-F,R。靶标基因为GhCOR27
COR27-R	AGTTGGTTTGAGTCAACTCAggaggccttctagagaattc	注释同CLA1-R,靶标基因为GhCOR27
GhU6-COR27-F	aagcgaaagaagcatTGAGTTGACTCAAACCAACT	注释同GhU6-CLA1-F,R,靶标基因为GhCOR27
PGT-COR27-F	caaagcaccagtggtctagtTGAGTTGACTCAAACCAACT	注释同PGTR-CLA1-F,靶标基因为GhCOR27

引物名称 Primers name	引物序列 Primer sequence(5'-3')	注释 Annotation
GhCLA1-F	CACGGTAACATACAGAATAAGCC	扩增包含CLA1靶序列的基因组片段
GhCLA1-R	ACGTCATGTACGACCTGTTGC	扩增包含CLA1靶序列的基因组片段
GhCOR27-F	CTCCGTCTTTGCATCCTC	扩增包含GhCOR27靶序列的基因组片段
GhCOR27-R	ATCAACCCTCTAGTTTCC	扩增包含GhCOR27靶序列的基因组片段

引物名称 Primers name	引物序列 Primer sequence(5'-3')	注释 Annotation
GhCLA1-F	CACGGTAACATACAGAATAAGCC	扩增包含CLA1靶序列的基因组片段
GhCLA1-R	ACGTCATGTACGACCTGTTGC	扩增包含CLA1靶序列的基因组片段
GhCOR27-F	CTCCGTCTTTGCATCCTC	扩增包含GhCOR27靶序列的基因组片段
GhCOR27-R	ATCAACCCTCTAGTTTCC	扩增包含GhCOR27靶序列的基因组片段

投递系统 Delivery system	靶标基因 Targeted gene	突变体数量/ 样本数量 No. of mutations No. of samples	突变率 Mutation rate (%)
TRV-AtU6::sgRNA	sgRNA-GhCLA1	13/20	65.0
TRV-AtU6::PTG-sgRNA		14/20	70.0
TRV-GhU6::sgRNA		20/25	80.0
TRV-GhU6::PTG-sgRNA		17/21	80.9

Efficient screening of sgRNA mediated by CRISPR/Cas9 via TRV viral vectors in cotton

利用TRV病毒载体在棉花中高效筛选CRISPR/Cas9介导的sgRNA

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投递系统 Delivery system	靶标基因 Targeted gene	突变体数量/ 样本数量 No. of mutations/ No. of samples	突变率 Mutation rate (%)
TRV-AtU6::sgRNA	sgRNA-GhCOR27	13/20	65.0
TRV-AtU6::PTG-sgRNA		14/20	70.0
TRV-GhU6::sgRNA		16/20	80.0
TRV-GhU6::PTG-sgRNA		17/20	85.0

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