DOI: 10.57760/sciencedb.26626
title: hfCas12Max-Mediated Targeted Integration at Accessible Chromatin Regions with a Novel UCOE Enhances Stable Recombinant Lactoferrin Expression
title: hfCas12Max-Mediated Targeted Integration at Accessible Chromatin Regions with a Novel UCOE Enhances Stable Recombinant Lactoferrin Expression

description: Transgenic production of recombinant proteins in livestock milk shows commercial potential, but stable transgene expression is often hindered by transgene silencing and poor integration site selection. This study used ATAC-seq to analyze chromatin accessibility in goat mammary epithelial cells, identifying 15 highly accessible sites. Validation through dual luciferase reporter assays and flow cytometry confirmed stable transgene expression at three of these sites. Additionally, we discovered a novel goat-derived ubiquitous chromatin-opening element (UCOE) in the SF3B1-COQ10B intergenic region, characterized by high GC content (65%) and CpG islands. This UCOE improved CRISPR/hfCas12Max-mediated transgene integration efficiency and stabilized human lactoferrin (hLTF) expression in GMECs. Notably, hfCas12Max was more effective than CRISPR/Cas9 for integrating large DNA fragments at specific loci. We used hfCas12Max to integrate the UCOE-hLTF cassette into the ROSA26 locus, creating transgenic goat lambs via somatic cell nuclear transfer without off-target effects. Our research establishes a chromatin accessibility-guided CRISPR/hfCas12Max gene-editing pipeline, highlights the pivotal role of UCOE elements containing CpG islands in overcoming transgene silencing, and provides a translational framework for high-performance mammary bioreactors. These findings enhance our understanding of the interplay between chromatin architecture, regulatory elements, and transgene expression in livestock, offering resources for precision breeding of lactoferrin-enriched dairy goats.
description: Transgenic production of recombinant proteins in livestock milk shows commercial potential, but stable transgene expression is often hindered by transgene silencing and poor integration site selection. This study used ATAC-seq to analyze chromatin accessibility in goat mammary epithelial cells, identifying 15 highly accessible sites. Validation through dual luciferase reporter assays and flow cytometry confirmed stable transgene expression at three of these sites. Additionally, we discovered a novel goat-derived ubiquitous chromatin-opening element (UCOE) in the SF3B1-COQ10B intergenic region, characterized by high GC content (65%) and CpG islands. This UCOE improved CRISPR/hfCas12Max-mediated transgene integration efficiency and stabilized human lactoferrin (hLTF) expression in GMECs. Notably, hfCas12Max was more effective than CRISPR/Cas9 for integrating large DNA fragments at specific loci. We used hfCas12Max to integrate the UCOE-hLTF cassette into the ROSA26 locus, creating transgenic goat lambs via somatic cell nuclear transfer without off-target effects. Our research establishes a chromatin accessibility-guided CRISPR/hfCas12Max gene-editing pipeline, highlights the pivotal role of UCOE elements containing CpG islands in overcoming transgene silencing, and provides a translational framework for high-performance mammary bioreactors. These findings enhance our understanding of the interplay between chromatin architecture, regulatory elements, and transgene expression in livestock, offering resources for precision breeding of lactoferrin-enriched dairy goats.