棉花学报2025，Vol.37Issue(2)：153-164,12.DOI:10.11963/cs20250005
棉花基因编辑标记基因GhOMT1和GhPGF高效sgRNA的筛选
Screening of highly efficient sgRNA for gene editing marker genes GhOMT1 and GhPGF in cotton
摘要
Abstract
[Objective]This study aimed to screen the single-guide RNA(sgRNA)for gene editing of GhOMT1(encoding flavonoid 3-O-methyltransferase)and GhPGF(related to gland formation)in cotton,laying the foundation for rapid detection of mutant plants.[Methods]Based on the Cas9-overexpressing cotton Jin668,sgRNAs targeting GhOMT1 and GhPGF genes were designed.The editing efficiency and specificity of the sgRNAs were detected by using the cotton leaf crumple virus(CLCrV)vector-mediated gene editing system and high-throughput tracking of mutations(Hi-TOM)technology.[Results]GhOMT1-sgRNA2 and GhPGF-sgRNA induced mutations at the target sites of GhOMT1 and GhPGF,respectively.Hi-TOM sequencing revealed that 12 out of 15 cotton plants transformed with GhOMT1-sgRNA2 exhibited mutations,with a mutation efficiency of 80％.The editing efficiency in single plants ranged from 7.90％to 43.72％for the A subgenome and from 9.60％to 56.32％for the D subgenome.The mutation efficiency of GhPGF-sgRNA was 80％,with an editing efficiency ranging from 10.23％to 30.27％.No off-target effects were observed at the three potential off-target sites of GhOMT1-sgRNA2,and GhPGF-sgRNA had no potential off-target sites in the coding regions of the genome,indicating the high specificity of these sgRNAs.[Conclusion]One sgRNA targeting GhOMT1 and one sgRNA targeting GhPGF were screened using the CLCrV-mediated gene editing system.关键词
棉花/标记基因/GhOMT1/GhPGF/棉花叶皱缩病毒/sgRNA/特异性Key words
cotton/marker genes/GhOMT1/GhPGF/cotton leaf crumple virus/sgRNA/specificity引用本文复制引用
郭浩猛,代培红,张锦恩,张国帅,雷建峰,刘晓东..棉花基因编辑标记基因GhOMT1和GhPGF高效sgRNA的筛选[J].棉花学报,2025,37(2):153-164,12.基金项目
国家自然科学基金(32301282) （32301282）
新疆维吾尔自治区自然科学基金青年基金(2022D01B23) （2022D01B23）
新疆维吾尔自治区"天山英才"培养计划"三农"骨干人才-现代农牧业发展高层次人才项目(2024SNGGGCC014) （2024SNGGGCC014）
