2. Academic Validation
  3. hfCas12Max-mediated targeted integration at accessible chromatin regions with a goat-derived UCOE enhances stable recombinant lactoferrin expression

hfCas12Max-mediated targeted integration at accessible chromatin regions with a goat-derived UCOE enhances stable recombinant lactoferrin expression

  • J Genet Genomics. 2025 Nov 17:S1673-8527(25)00298-X. doi: 10.1016/j.jgg.2025.11.008.
Zhenliang Zhu 1,  Jing Han 2,  Jianglin Zhao 2,  Yangyi Jian 3,  Yong Zhang 3,  Yayi Liu 2,  Peipei Bian 4,  Zhenyu Wei 4,  Xiaodan Ma 5,  Rui Feng 5,  Yong Zhang 6,  Yuanpeng Gao 7,  Jun Liu 8
Affiliations

Affiliations

  • 1 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China; National Center of Technology Innovation for Dairy, Hohhot, Inner Mongolia 010000, China.
  • 2 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China.
  • 3 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China.
  • 4 College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
  • 5 Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China; National Center of Technology Innovation for Dairy, Hohhot, Inner Mongolia 010000, China.
  • 6 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China; National Center of Technology Innovation for Dairy, Hohhot, Inner Mongolia 010000, China. Electronic address: [email protected].
  • 7 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: [email protected].
  • 8 College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China; National Center of Technology Innovation for Dairy, Hohhot, Inner Mongolia 010000, China. Electronic address: [email protected].
PMID: 41260248 DOI: 10.1016/j.jgg.2025.11.008
Abstract

Stable transgene expression in the mammary gland is crucial for recombinant protein production in livestock, yet it is frequently hampered by transgene silencing and random integration. To address this, we profile chromatin accessibility in goat mammary epithelial cells (GMECs) using ATAC-seq and identify 15 highly accessible genomic regions. Three of these regions are confirmed to support stable transgene expression. Notably, we identify a goat-derived ubiquitous chromatin opening element (UCOE) in the SF3B1-COQ10B intergenic region, with a high GC content (65%) and CpG island enrichment. This UCOE improves hfCas12Max-mediated integration of large DNA fragments and maintains high-level expression of human lactoferrin (hLTF) in GMECs. Subsequently, we precisely integrate the UCOE-hLTF cassette into the highly accessible loci and generate a transgenic goat via somatic cell nuclear transfer, without detectable off-target effects. Our pipeline, which integrates chromatin accessibility profiling, UCOE discovery, and precision editing, demonstrates the role of CpG island-containing UCOEs in preventing transgene silencing. The study provides valuable tools for enhancing recombinant protein production and supports the breeding of dairy goats for milk with high lactoferrin content, while advancing the understanding of the interactions between chromatin, regulatory elements, and transgenes in molecular breeding.

Keywords

ATAC-seq; CpG island; Goat mammary gland bioreactor; Integration sites; Ubiquitous chromatin-opening element (UCOE); hfCas12Max.

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