Precise Editing of the FGF18 Gene in Sheep Fibroblasts Using the AncBE4max System

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

Abstract

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

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.

Figures/Tables 10

References 33

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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

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

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

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