靶向敲除棉花GhAGL16高效sgRNA的筛选

doi:10.3864/j.issn.0578-1752.2024.06.001

Abstract

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

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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Figures/Tables 9

Table 1

Primer sequences used in this study"

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

Table 1

Fig. 1

Fig.2

Fig. 3

Fig. 4

Fig. 5

Table 2

Fig. 6

Table 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

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

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