靶向敲除棉花GhAGL16高效sgRNA的筛选

doi:10.3864/j.issn.0578-1752.2024.06.001

中国农业科学 ›› 2024, Vol. 57 ›› Issue (6): 1023-1033.doi: 10.3864/j.issn.0578-1752.2024.06.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

靶向敲除棉花GhAGL16高效sgRNA的筛选

雷建峰¹(), 尤扬子², 张锦恩², 代培红², 于莉², 杜正阳¹, 李月², 刘晓东²()

¹ 新疆农业大学农学院/棉花教育部工程研究中心，乌鲁木齐 830052
² 新疆农业大学生命科学学院，乌鲁木齐 830052

收稿日期:2023-09-04 接受日期:2023-11-03 出版日期:2024-03-16 发布日期:2024-03-25
通信作者:
刘晓东，E-mail：xiaodongliu75@aliyun.com
联系方式: 雷建峰，E-mail：kyleijianfeng@163.com。
基金资助:
国家自然科学基金(32301282); 国家自然科学基金(32160494); 新疆维吾尔自治区自然科学基金(2022D01B23); 自治区高校基本科研业务费科研项目(XJEDU2022J007); 新疆农业大学作物学重点学科发展基金(XNCDKY2021002); 新疆农业大学生物学创新团队开放课题(ITB202106); 新疆维吾尔自治区大学生创新创业训练计划(S202210758037)

Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton

LEI JianFeng¹(), YOU YangZi², ZHANG JinEn², DAI PeiHong², YU Li², DU ZhengYang¹, LI Yue², LIU XiaoDong²()

¹ College of Agronomy, Xinjiang Agricultural University/Cotton Engineering Research Center, Ministry of Education, Urumqi 830052
² College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052

Received:2023-09-04 Accepted:2023-11-03 Published:2024-03-16 Online:2024-03-25

摘要/Abstract

摘要：

【目的】 AGL16是棉花MADS-box基因家族中一个重要的转录负调控因子，在抵御干旱和盐胁迫过程中发挥着重要作用。利用病毒介导的基因编辑技术（virus-induced gene editing，VIGE）筛选靶向敲除棉花GhAGL16的sgRNAs，并验证这些sgRNAs的特异性，为定向创制棉花agl16突变体奠定重要基础。【方法】根据在棉花YZ-1中A亚组和D亚组上实际克隆GhAGL16的基因组序列，预测了3个能靶向敲除GhAGL16的sgRNAs；基于棉花叶皱缩病毒（cotton leaf crumple virus，CLCrV）介导的VIGE系统，构建3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体；利用qPCR检测Cas9超表达（Cas9 over-expression，Cas9-OE）植株中Cas9的表达量，确定Cas9是否稳定遗传表达；将3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体分别转化Cas9-OE棉花子叶，并通过PCR/RE方法检测3个靶点的突变情况；利用生物信息学方法分析3个GhAGL16-sgRNAs的二级结构；对突变植株进行Hi-TOM高通量测序，明确基因编辑效率。同时，对转化GhAGL16-sgRNA2的突变植株进行脱靶率鉴定，检测基因编辑的特异性。【结果】成功构建了3个能同时靶向敲除GhAGL16-A亚组和GhAGL16-D亚组序列的sgRNAs。对不同Cas9-OE植株中Cas9表达量检测结果表明，Cas9在不同Cas9-OE棉株中稳定表达。用3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体转化Cas9-OE棉花子叶，PCR/RE突变检测结果表明，GhAGL16-sgRNA2可有效用于GhAGL16的靶向敲除，在棉花A亚组和D亚组靶位点上出现了不同碱基缺失的突变类型，而GhAGL16-sgRNA1和GhAGL16-sgRNA3是2个无效sgRNA。3个GhAGL16-sgRNAs的二级结构分析结果表明，GhAGL16-sgRNA1和GhAGL16-sgRNA3可能存在引导序列容易与其他序列发生配对并且不易解链的现象，干扰了引导序列对目标位点的识别，导致sgRNA无效。进一步量化GhAGL16-sgRNA2对GhAGL16的编辑效率，对每一株转化CLCrV-AtU6-26::GhAGL16-sgRNA2的Cas9-OE植株突变检测结果表明，在转化的9株Cas9-OE植株中有6株发生了突变，突变效率为66.67%。此外，Hi-TOM高通量测序结果表明，GhAGL16-sgRNA2对GhAGL16的编辑效率为13.69%—54.42%。脱靶率鉴定结果表明，预测的4个潜在脱靶位点都没有发现脱靶现象，说明GhAGL16-sgRNA2不仅具有高效的基因编辑效率，而且具有专一的基因编辑特异性。【结论】利用CLCrV介导的VIGE系统转化Cas9-OE棉花筛选获得1个能高效敲除GhAGL16的sgRNA，为定向创制棉花agl16突变体提供了理想的sgRNA。

关键词: 棉花, GhAGL16, sgRNA, 突变体, VIGE, 脱靶

Abstract:

【Objective】 As an important negative transcriptional regulator in cotton MADS-box gene family, AGL16 plays an important role in resisting drought and salt stress. Virus-induced gene editing (VIGE) was used to screen sgRNAs that knockout the cotton GhAGL16 and verify the specificity of these sgRNAs, which laid a foundation for the creation of cotton agl16 mutants.【Method】 Three sgRNAs could knockout GhAGL16 were predicted based on the actual GhAGL16 genomic sequence cloned on subgroup A and D in cotton YZ-1; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were constructed based on the cotton leaf crumple virus (CLCrV)-mediated VIGE system; The expression of Cas9 in Cas9 over-expression (Cas9-OE) plants was detected by qPCR to determine whether Cas9 was stably genetically expressed; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were transformed respectively into Cas9-OE cotton cotyledons and detected the mutations of the three targets by PCR/RE; The secondary structures of three GhAGL16-sgRNAs were analyzed by bioinformatics; Hi-TOM high-throughput sequencing was performed on mutant plants to determine the efficiency of gene editing. Meanwhile, the off-target rate of GhAGL16-sgRNA2 mutant plants were identified to detect the specificity of gene editing.【Result】 Three sgRNAs capable of simultaneously knocking out GhAGL16-A and D subgroups were successfully constructed. The detection results of Cas9 expression showed that Cas9 was stably expressed in different Cas9-OE cotton plants. PCR/RE mutation detection results showed that GhAGL16-sgRNA2 could be effectively used for the knockout of GhAGL16. Different mutation types with base deletions appeared at the target sites of cotton subgroups A and D, while GhAGL16-sgRNA1 and GhAGL16-sgRNA3 were two invalid sgRNAs. The secondary structure analysis results of three GhAGL16-sgRNAs indicated that GhAGL16-sgRNA1 and GhAGL16-sgRNA3 might have a phenomenon that the guide sequence was easy to pair with other sequences and difficult to unwind, which interfered with the recognition of the target site by the guide sequences and lead to the invalid sgRNA. To further quantify the editing efficiency of GhAGL16-sgRNA2 on GhAGL16, the mutation detection results of each Cas9-OE plant transformed with CLCrV-AtU6-26::GhAGL16-sgRNA2 showed that six of the nine Cas9-OE plants were mutated, with a mutation efficiency of 66.67%. In addition, Hi-TOM high-throughput sequencing results showed that the editing efficiency of GhAGL16-sgRNA2 for GhAGL16 was 13.69%-54.42%. The off-target identification results showed that no off-target phenomenon was detected at the four predicted off-target sites, indicating that GhAGL16-sgRNA2 not only has high gene editing efficiency, but also has specific gene editing specificity.【Conclusion】 A sgRNA that can effectively knocking out the GhAGL16 was obtained by transforming Cas9-OE cotton using the CLCrV-mediated VIGE system, providing an ideal sgRNA for creating cotton agl16 mutants.

Key words: cotton, GhAGL16, sgRNA, mutant, VIGE, off-target

雷建峰, 尤扬子, 张锦恩, 代培红, 于莉, 杜正阳, 李月, 刘晓东. 靶向敲除棉花GhAGL16高效sgRNA的筛选[J]. 中国农业科学, 2024, 57(6): 1023-1033.

LEI JianFeng, YOU YangZi, ZHANG JinEn, DAI PeiHong, YU Li, DU ZhengYang, LI Yue, LIU XiaoDong. Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton[J]. Scientia Agricultura Sinica, 2024, 57(6): 1023-1033.

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图/表 9

表1

本研究中使用的引物序列"

引物Primer	序列Sequence (5′-3′)	目的Destination
GhAGL16-sgRNA1F	GATTGCTACGATTTCGCTAGCACC	构建AtU6-26::GhAGL16-sgRNA1 Construction of AtU6-26::GhAGL16-sgRNA1
GhAGL16-sgRNA1R	AAACGGTGCTAGCGAAATCGTAGC
GhAGL16-sgRNA2F	GATTGCTGCTGTGGCTGGCTTAGC	构建AtU6-26::GhAGL16-sgRNA2 Construction of AtU6-26::GhAGL16-sgRNA2
GhAGL16-sgRNA2R	AAACGCTAAGCCAGCCACAGCAGC
GhAGL16-sgRNA3F	GATTGGTAAAAGATTTACAGAATT	构建AtU6-26::GhAGL16-sgRNA3 Construction of AtU6-26::GhAGL16-sgRNA3
GhAGL16-sgRNA3R	AAACAATTCTGTAAATCTTTTACC
M1-GhAGL16F	AAATAACCTCCATTGCATTTCTCTT	PCR扩增涵盖GhAGL16靶位点1区域（509 bp） PCR amplification contains the GhAGL16 gene target site 1 region (509 bp)
M1-GhAGL16R	CAACATTTTACCGTCACATTTAATC
M2-GhAGL16F	TCCGTATGAAGAAGGTTTGAAAATT	PCR扩增涵盖 GhAGL16靶位点2区域（797 bp） PCR amplification contains the GhAGL16 gene target site 2 region (797 bp)
M2-GhAGL16R	GAGAAGCATAACTTTTGGAACC
M3-GhAGL16F	CCATATAAATCACTATATCAAACGTG	PCR扩增涵盖 GhAGL16靶位点3区域（682 bp） PCR amplification contains the GhAGL16 gene target site 3 region (682 bp)
M3-GhAGL16R	ATTTTCAAACCTTCTTCATACGGA
HiT-GhAGL16F	GGAGTGAGTACGGTGTGCCAAGGGATGTCAATGGTGCA	高通量测序检测GhAGL16编辑效率 Detection of GhAGL16 gene editing efficiency by high-throughput sequencing
HiT-GhAGL16R	GAGTTGGATGCTGGATGGTGGAGATCAGTTTGTGGCAA
Q-GhUBQ7F	GAAGGCATTCCACCTGACCAAC	PCR扩增GhUBQ7部分片段 PCR amplification of partial fragments of GhUBQ7 gene
Q-GhUBQ7R	CTTGACCTTCTTCTTCTTGTGCTTG
Q-Cas9F	GTCATTACGGACGAGTACAAG	qPCR分析Cas9 mRNA表达量 qPCR analysis of Cas9 mRNA expression
Q-Cas9R	AGGTAGCAGATCCGATTCTTT
Gh_A06G030400-F	GGAGTGAGTACGGTGTGCGAACTTTCTGGATTGGGTGTAA	高通量测序检测Gh_A06G030400 Detection of Gh_A06G030400 gene by high-throughput sequencing
Gh_A06G030400-R	GAGTTGGATGCTGGATGGATTGCAGTCCCAAGTTTGTAG
Gh_A06G081500-F	GGAGTGAGTACGGTGTGCGCCAATCCAAATATGTCCAGC	高通量测序检测Gh_A06G081500 Detection of Gh_A06G081500 gene by high-throughput sequencing
Gh_A06G081500-R	GAGTTGGATGCTGGATGGTTAGCCACACCGTCTTCCA
Gh_A08G005400-F	GGAGTGAGTACGGTGTGCTTACTTCCAGAGCAGACAC	高通量测序检测Gh_A08G005400 Detection of Gh_A08G005400 gene by high-throughput sequencing
Gh_A08G005400-R	GAGTTGGATGCTGGATGGGCATATGGTCGACTTAGCA
Gh_A09G158200-F	GGAGTGAGTACGGTGTGCAGTGGTGCAAAAGGGGAG	高通量测序检测Gh_A09G158200 Detection of Gh_A09G158200 gene by high-throughput sequencing
Gh_A09G158200-R	GAGTTGGATGCTGGATGGCTTAATGAGATAGTTGCTGGC

表1

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表2

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表3

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基因组<BOLD>G</BOLD>enome	植株编号 Plant number	样本读取数量 No. of sample reads	突变比例 Mutation ratio (%)	突变类型 Mutation type	突变序列 Mutations in the sequence	突变位点（sgRNA2） Mutation site (sgRNA2, 5′-3′)
A亚组 Subgroup A	WT	403	100	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#2	584	87.29	WT	-	CCAGCTAAGCCAGCCACAGCAGC
		39	5.83	2D	CT	CCAG- -AAGCCAGCCACAGCAGC
		25	3.74	2D	AA	CCAGCT- -GCCAGCCACAGCAGC
		21	3.14	1D	A	CCAGCT-AGCCAGCCACAGCAGC
	#6	921	79.60	WT	-	CCAGCTAAGCCAGCCACAGCAGC
		127	10.98	2D	CT	CCAG- -AAGCCAGCCACAGCAGC
		71	6.14	1D	A	CCAGCT-AGCCAGCCACAGCAGC
		38	3.28	4D	AAGC	CCAGCT- - - -CAGCCACAGCAGC
	#7	569	90.61	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#7	59	9.39	2D	CT	CCAG- -AAGCCAGCCACAGCAGC
	#8	485	84.79	WT	-	CCAGCTAAGCCAGCCACAGCAGC
		59	10.31	SNP	A<G	CCAGCTAGGCCAGCCACAGCAGC
		28	4.90	3D	AAG	CCAGCT- - -CCAGCCACAGCAGC
	#9	412	91.76	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#9	37	8.24	2D	CT	CCAG- -AAGCCAGCCACAGCAGC
D亚组 Subgroup D	WT	531	100	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#4	500	96.71	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#4	17	3.29	3D	AAG	CCAGCT- - -CCAGCCACAGCAGC
	#6	64	65.98	WT	-	CCAGCTAAGCCAGCCACAGCAGC
		18	18.56	2D	CT	CCAG- -AAGCCAGCCACAGCAGC
		15	15.46	5D	AAGCC	CCAGCT- - - - -AGCCACAGCAGC
	#8	408	86.99	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#8	61	13.01	1D	A	CCAGCT-AGCCAGCCACAGCAGC
	#9	434	94.55	WT	-	CCAGCTAAGCCAGCCACAGCAGC
	#9	25	5.45	1D	A	CCAGCT-AGCCAGCCACAGCAGC

潜在脱靶位点的序列 Sequence of the putative off-target site (5′-3′)	匹配碱基数量（包括PAM） No. of matching bases (include PAM)	基因 Gene	区域 Region	样本读取数量 No. of sample reads
GTTGCTGTGGCTGGCACAGCTGG	20	Gh_A06G030400	CDS	976
GCTGCTGTGACTGGCATTGATGG	19	Gh_A06G081500	CDS	965
GCTGCTGTGGCTGTTCAAGCTGG	19	Gh_A08G005400	CDS	1021
GCTGCTGCTGCTGGCTATGCAGG	18	Gh_A09G158200	CDS	855

靶向敲除棉花GhAGL16高效sgRNA的筛选

Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton

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