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Published on: September 25, 2019
Precise Editing of chNHE1 Gene via CRISPR/Cas9 Generates ALV-J-Resistant Chicken Primordial Germ Cell
Xinyi Zhou1, Ruyu Liao1, Min Tan1
1College of Animal Science and Technology, Southwest University, Chongqing 400715, China.
Abstract:
Avian leukosis virus subgroup J (ALV-J), an α-retrovirus, mediates infection by binding to the host-specific receptor chNHE1 (chicken sodium-hydrogen exchanger type 1), leading to immunosuppression and tumorigenesis, which severely threatens the sustainable development of the poultry industry. Studies have shown that the tryptophan residue at position 38 (W38) of the chNHE1 protein is the critical site for ALV-J infection. In this study, we employed the CRISPR/Cas9 system to construct a lentiviral vector targeting the W38 site of chNHE1, transfected it into chicken primordial germ cells (PGCs), and validated its antiviral efficacy through ALV-J infection assays, successfully establishing an in vitro gene-editing system for chicken PGCs. The constructed dual lentiviral vector efficiently targeted the W38 site. PGCs isolated from 5.5- to 7-day-old chicken embryos were suitable for in vitro gene editing. Stable fluorescence expression was observed within 24-72 h post-transfection, confirming high transfection efficiency. ALV-J challenge tests demonstrated that no viral env gene expression was detected in transfected PGCs at 48 h or 72 h post-infection, while high env expression was observed in control groups. After 7 days of infection, p27 antigen ELISA tests were negative in transfected groups but positive in controls, indicating that W38-deleted PGCs exhibited strong resistance to ALV-J. This study successfully generated ALV-J-resistant gene-edited PGCs using CRISPR/Cas9 technology, providing a novel strategy for disease-resistant poultry breeding and advancing avian gene-editing applications.
Insights
CRISPR/Cas9 gene editing created chicken primordial germ cells resistant to Avian leukosis virus subgroup J (ALV-J) by modifying the chNHE1 receptor. This breakthrough offers a new strategy for developing disease-resistant poultry and advancing avian gene editing.
Area of Science:
- * Molecular Biology
- * Genetics
- * Animal Science
Background:
- * Avian leukosis virus subgroup J (ALV-J) causes immunosuppression and tumors in poultry, impacting the poultry industry.
- * ALV-J infection relies on binding to the chicken sodium-hydrogen exchanger type 1 (chNHE1) receptor, specifically at the tryptophan residue 38 (W38).
Purpose of the Study:
- * To develop an in vitro gene-editing system in chicken primordial germ cells (PGCs) to confer resistance against ALV-J.
- * To utilize CRISPR/Cas9 technology to target and modify the W38 site of the chNHE1 receptor in chicken PGCs.
Main Methods:
- * Construction of a dual lentiviral vector for CRISPR/Cas9 delivery targeting the W38 site of chNHE1.
- * Transfection of chicken PGCs with the lentiviral vector and validation of gene editing efficiency.
- * ALV-J infection assays on gene-edited and control PGCs, including env gene expression analysis and p27 antigen detection.
Main Results:
- * The CRISPR/Cas9 system efficiently targeted the W38 site in chicken PGCs, with high transfection rates observed.
- * Gene-edited PGCs showed no detectable viral env gene expression post-ALV-J infection at 48 and 72 hours.
- * ELISA tests confirmed the absence of p27 antigen in edited PGCs after 7 days of infection, indicating resistance.
Conclusions:
- * Successfully generated ALV-J-resistant gene-edited chicken PGCs using CRISPR/Cas9 technology.
- * The W38-modified chNHE1 receptor effectively blocks ALV-J entry, demonstrating significant antiviral efficacy.
- * This study provides a novel strategy for disease-resistant poultry breeding and advances applications in avian gene editing.
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