Cotton Science

Screening of highly efficient sgRNA for gene editing marker genes GhOMT1 and GhPGF in cotton

Guo Haomeng, Dai Peihong, Zhang Jin’en, Zhang Guoshuai, Lei Jianfeng, Liu Xiaodong

Cotton Science, 2025, 37(2): 153-164. DOI: 10.11963/cs20250005

植株编号 Plant number	亚基因组（编辑效率） Subgenome (editing efficiency)	突变类型 Mutation type	序列 Sequence	占比 Ratio/%
#9	A (43.72%)	WT	AACGCCGTGGTACTTCCAATGG	56.28
		SNP, 1D	AACGCCGTGATACTT-CAATGG	8.60
		1D	AACGCCGTGGTACTTC-AATGG	6.57
		SNP, 1I	AACGCCGTGATACTTCTCAATGG	6.40
		SNP, 4D	AACGCCGTGATA----CAATGG	4.70
		1I	AACGCCGTGGTACTTCACAATGG	3.74
		1I	AACGCCGTGGTACTTCTCAATGG	3.51
		3D	AACGCCGTGGTA---CCAATGG	3.23
		SNP, 3D	AACGCCGTGATA---CCAATGG	2.43
		8D	AACGCCGTG--------AATGG	1.76
		SNP, 1I	AACGCCGTGATACTTCACAATGG	1.53
		11D	AACG-----------CCAATGG	1.25
	D (47.80%)	WT	AACGCCGTGGTACTTCCAATGG	52.20
		1D	AACGCCGTGGTACTT-CAATGG	13.10
		1I	AACGCCGTGGTACTTCGCAATGG	7.48
		3D	AACGCCGTGGTA---CCAATGG	6.96
		4D	AACGCCGTGGTA----CAATGG	5.09
		1I	AACGCCGTGGTACTTCACAATGG	4.57
		6D	AACGCCGTGG------CAATGG	3.22
		8D	AACGCCGTGGTACT--------	2.70
		1I	AACGCCGTGGTACTTCTCAATGG	2.70
		12D	AACG------------CAATGG	1.98
#12	A (35.63%)	WT	AACGCCGTGGTACTTCCAATGG	64.37
		SNP, 1D	AACGCCGTGATACTT-CAATGG	6.63
		SNP, 1I	AACGCCGTGATACTTCTCAATGG	6.46
		1D	AACGCCGTGGTACTT-CAATGG	5.78
		1I	AACGCCGTGGTACTTCTCAATGG	3.96
		SNP, 4D	AACGCCGTGATA----CAATGG	3.91
		3D	AACGCCGTGGTA---CCAATGG	3.79
		1I	AACGCCGTGGTACTTCACAATGG	3.68
		3D	AACGCCGTGGTACTT---ATGG	1.42
	D (56.32%)	WT	AACGCCGTGGTACTTCCAATGG	43.68
		5D	AACGCCGTGGTACT-----TGG	13.45
		1D	AACGCCGTGGTACTT-CAATGG	7.64
		3D	AACGCCGTGGTA---CCAATGG	6.16
		1I	AACGCCGTGGTACTTCACAATGG	5.02
		1I	AACGCCGTGGTACTTCTCAATGG	4.79
		4D	AACGCCGTGGTA----CAATGG	4.56
		2D	AACGCCGTGGTACT--CAATGG	4.56
		6D	AACGCCGTGG------CAATGG	3.53
		5D	AACGCCGTGGT-----CAATGG	2.51
		14D	AACGCC--------------GG	2.05
		11D	AACGCCG-----------ATGG	2.05

Table 1 Analysis of mutation patterns in plants with high editing efficiency of GhOMT1

Other figure/table from this article

Fig. 1 Sequencing chromatogram of target sites of GhOMT1 and GhPGF A: Comparison of sequencing chromatogram of target sites of GhOMT1-sgRNA2 in wild type (WT) and Cas9-OE plants transformed with CLCrV-AtU6-26::GhOMT1-sgRNA viral vectors. B: Comparison of sequencing chromatogram of target sites of GhPGF-sgRNA in wild type (WT) and Cas9-OE plants transformed with CLCrV-AtU6-26::GhPGF-sgRNA viral vectors.
Table 2 Analysis of mutation patterns in plants with high and low editing efficiency of GhPGF
Table 3 Relationship between viral accumulation and gene editing efficiency
Fig. 2 Analysis of the predicted secondary structures of GhOMT1-sgRNAs and GhPGF-sgRNA A-H: Secondary structures of GhOMT1-sgRNA1 to GhOMT1-sgRNA8. I: Secondary structure of GhPGF-sgRNA. RAR: retinoic acid receptors.
Fig. 3 Sequence alignment analysis of the 3 potential off-target sites for GhOMT1-sgRNA2 PAM: protospacer adjacent motif. Off-score: off-target effect score. Consensus: consistent base sequence.
Table 4 Detection and analysis of the potential off-target sites for GhOMT1-sgRNA2