基于AncBE4max系统精准编辑绵羊成纤维细胞FGF18基因

doi:10.13560/j.cnki.biotech.bull.1985.2025-0454

摘要/Abstract

摘要：

目的成纤维细胞生长因子18（FGF18）作为毛囊周期的关键调控分子，对毛发生长、毛囊发育具有核心调控作用，而在绵羊等家畜中，其调控羊毛生长的功能与分子机制尚未明晰。基于此，建立一种精准的绵羊成纤维细胞生长因子18（FGF18）单碱基突变系统，利用单碱基编辑系统对绵羊FGF18基因进行定点编辑，探索该技术在农牧业基因改良中的应用潜力，为未来提高羊毛产量提供理论依据。方法根据绵羊成纤维细胞FGF18基因的第3、4外显子序列设计并合成了3个单导向RNA（single guide，sgRNA）及其互补链，将退火连接形成的sgRNA分别克隆至pGL3-U6-sgRNA-PGK-puromycin表达质粒中。通过电转染方式，将含有特异性sgRNA的U6表达质粒与AncBE4max质粒共转染至绵羊成纤维细胞，转染72 h后对细胞进行测序验证。结果 PCR扩增的FGF18基因片段经T-A克隆后，测序结果表明在FGF18基因的第3、第4外显子内成功引入了终止密码子。通过筛选和鉴定，成功获得了2个可有效定点编辑绵羊成纤维细胞FGF18基因的sgRNA（sg1和sg3），其编辑效率分别为13.8%和36.4%。结论建立的基于AncBE4max系统的绵羊成纤维细胞FGF18基因单碱基编辑系统，可实现外显子区域终止密码子的精准引入，并筛选出sgRNA-sg1和sgRNA-sg3两个有效编辑靶点。

关键词: AncBE4max, 成纤维细胞生长因子18, 基因编辑, 单碱基编辑, 绵羊成纤维细胞

Abstract:

Objective Fibroblast growth factor 18 (FGF18) is a critical regulator of the hair follicle cycle and plays a central role in hair growth and follicle development. However, its function and molecular mechanisms in regulating wool growth remain poorly understood in livestock such as sheep. Therefore, we established an efficient and precise base-editing system for sheep FGF18 to achieve site-specific editing of the FGF18 gene. This study investigates the potential applications of this technology for genetic improvement in agriculture, providing a theoretical foundation for enhancing wool production in the future. Method Three single guide RNAs and their complementary strands were designed and synthesized based on the sequences of exons 3 and 4 of the ovine FGF18 gene in fibroblasts. The annealed sgRNAs were cloned into the pGL3-U6-sgRNA-PGK-puromycin expression plasmid. The U6 expression plasmid containing specific sgRNA and AncBE4max plasmid were co-transfected into ovine fibroblasts via electroporation. Seventy-two hours after transfection, cells were subjected to sequencing verification. Result Results showed that after T-A cloning of the PCR-amplified FGF18 gene fragment, sequencing confirmed the successful introduction of stop codons into exon 3 and 4 of the FGF18 gene. Through screening and identification, two sgRNAs (sg1 and sg3) effectively mediating site-directed editing of the ovine FGF18 gene in fibroblasts were obtained, with editing efficiencies of 13.8% and 36.4%, respectively. Conclusion The established single-base editing system for the ovine fibroblast FGF18 gene, based on the AncBE4max system, enables the precise introduction of stop codons in exon regions. Additionally, two highly efficient editing targets, sgRNA-sg1 and sgRNA-sg3, are successfully screened.

Key words: AncBE4max, fibroblast growth factor 18, gene editing, base editing, sheep fibroblast cells

蒙亚琦, 王嵩, 杨鹏, 于航, 姚旭东, 郭延华, 唐红, 张译元, 王立民, 周平. 基于AncBE4max系统精准编辑绵羊成纤维细胞FGF18基因[J]. 生物技术通报, 2025, 41(10): 313-320.

MENG Ya-qi, WANG Song, YANG Peng, YU Hang, YAO Xu-dong, GUO Yan-hua, TANG Hong, ZHANG Yi-yuan, WANG Li-min, ZHOU Ping. Precise Editing of the FGF18 Gene in Sheep Fibroblasts Using the AncBE4max System[J]. Biotechnology Bulletin, 2025, 41(10): 313-320.

图/表 10

表1 sgRNA序列

Table 1 sgRNAs’ sequence

名称 Name	外显子区域 Exon region	引物序列 Primer sequence （5'-3'）
sg1	第三外显子	F：accgGGAGAACCAGACGCGGGCTC
sg1	第三外显子	R：aaacGAGCCCGCGTCTGGTTCTCC
sg2	第三外显子	F：accgACATCCAGGTCCTGGGCCGC
sg2	第三外显子	R：aaacGCGGCCCAGGACCTGGATGT
sg3	第四外显子	F：accgGGTAGTCAAGTCCGGATCAA
sg3	第四外显子	R：aaacTTGATCCGGACTTGACTACC

表2 PCR引物序列

Table 2 PCR primers' sequence

名称 Name	引物序列 Primer sequence （5'-3'）	退火温度 Annealing temperature （℃）	产物长度 Product size （bp）
E3-1	F：GCCAAGCAGGGCAGTTAC	57.9	562
E3-1	R：GGCATTGACCAGCAAGAGTA	57.9	562
E4-1	F：GAGTTGGGAACAGGTGTCA	55.6	588
E4-1	R：CTGCTAAGCAAGCCAGAGT	55.6	588

表3 脱靶位点具体信息

Table 3 Specific information of off-target site

名称

Name

脱靶序列

Off-target sequence

PCR引物序列

PCR primers’ sequence （5'-3'）

染色体位置

Chromosomal location

脱靶-1

GGACAGACAGAGGCGGGCTC

F：TGATGGATGTTGGCAGTGGT

R：GCTGACAGACGCCGTGGTTC

脱靶-2

CCAGGACCAGACGCAGGCTC

F：TTCCAGAGGAGGAGCATG

R：ACTCAGCGTGACAACAGATAG

图1 绵羊FGF18基因sgRNA靶区结构示意图

Fig. 1 Schematic diagram of the structure of the sgRNA targeting region of sheep FGF18 gene

图2 sgRNA质粒构建A：sgRNA-sg1质粒测序结果； B：sgRNA-sg2质粒测序结果； C：sgRNA-sg3质粒测序结果

Fig. 2 Construction of sgRNA plasmidA: sgRNA-sg1 plasmid sequencing result; B: sgRNA-sg2 plasmid sequencing result; C: sgRNA-sg3 plasmid sequencing result

图3 表达载体共转染在成纤维细胞中表达A：转染细胞明场； B：转染细胞GFP荧光

Fig. 3 Expression vectors co-transfected into fibroblasts for expressionA: Brightfield of transfected cells; B: GFP fluorescence of transfected cells

图4 sgRNA-sg1和sgRNA-sg3 PCR产物测序结果A：sgRNA-sg1 PCR测序结果； B：sgRNA-sg3 PCR测序结果。红色方框位置碱基为发生突变的位点

Fig. 4 Sequencing results of sgRNA-sg1 and sgRNA-sg3 PCR productsA: PCR sequencing results of sgRNA-sg1; B: PCR sequencing results of sgRNA-sg3. The base at the position of the red box is the site of mutation

表4 T-A克隆结果

Table 4 T-A cloning results

sgRNA名称

sgRNA name

靶位点序列信息

Target ID sequences （5′-3′）

突变类型

Mutation

突变比例

Mutation ratio （%）

sgRNA-sg1

GGAGAACCAGACGCGGGCTCGGG

GGAGAATTAGACGCGGGCTCGGG

GGAGAATCAGACGCGGGCTCGGG

GGAGAACCAGACGTGGGCTCGGG

C→T

34.5（10/29）

13.8（4/29）

17.2（5/29）

3.4（1/29）

sgRNA-sg3

GGTAGTCAAGTCCGGATCAAGGG

GGTAGTTAAGTCCGGATCAAGGG

GGTAG-------CGGATCAAGGG

C→T

C→T，-7 bp/fs

40.9（9/22）

36.4（8/22）

4.5（1/22）

图5 突变FGF18基因的蛋白结构的变化A：野生型-FGF18蛋白的3D模型； B：sgRNA-sg1突变体蛋白的3D模型； C：sgRNA-sg3突变体蛋白的3D模型

Fig. 5 Variation of protein structure of mutated FGF18 geneA: 3D model of wild-type-FGF18 protein; B: 3D model of mutant sgRNA-sg1 protein; C: 3D model of mutant sgRNA-sg3 protein

图6 预测脱靶位点测序结果A：脱靶-1 PCR测序结果；B：脱靶-2 PCR测序结果

Fig. 6 Sequencing-based prediction of off-target sitesA: PCR sequencing results of off-target-1; B: PCR sequencing results of off-target-2

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