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

Zhenliang Zhu; Jing Han; Jianglin Zhao; Yangyi Jian; Yong Zhang; Yayi Liu; Peipei Bian; Zhenyu Wei; Xiaodan Ma; Rui Feng; Yong Zhang; Yuanpeng Gao; Jun Liu

doi:10.1016/j.jgg.2025.11.008

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

doi: 10.1016/j.jgg.2025.11.008

a. College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi 712100, China;
b. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China;
c. National Center of Technology Innovation for Dairy, Hohhot, Inner Mongolia 010000, China;
d. College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China

Funds:

This work was supported by the Major Project on Agricultural Biological Breeding (2022ZD04014), the Shanxi Provincial Livestock and Poultry Breeding Industry "Two Chains" Integration Key Special Project "Cow Molecular Breeding and Rapid Breeding Key Technology" (2022GD-TSLD-46-0101), the Inner Mongolia Autonomous Region Unveils Marshalled Projects (2022JBGS0021), and the National Dairy Industry Technology Innovation Center Project Topics (2022-KYGG-3).

Received Date: 2025-06-18
Accepted Date: 2025-11-11
Rev Recd Date: 2025-11-11

Available Online: 2025-11-18

Abstract

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

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