Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9

Area of Science:

• * Biotechnology and Genetic Engineering
• * Xenotransplantation Research
• * Viral Eradication Strategies

Background:

• * Organ transplantation faces a critical shortage, driving interest in xenografts.
• * Porcine endogenous retroviruses (PERVs) pose a significant risk of cross-species infection in xenotransplantation.
• * Previous work quantified PERV presence in porcine cell lines (PK15) and primary fibroblasts.

Purpose of the Study:

• * To develop a method for inactivating PERVs in porcine cells.
• * To mitigate the risk of PERV transmission in pig-to-human xenotransplantation.
• * To provide the methodological foundation for eradicating PERVs in vitro.

Main Methods:

• * Utilized CRISPR-Cas9 gene editing technology.
• * Targeted and disrupted the catalytic center of the pol gene, crucial for viral replication.
• * Isolated and verified porcine cells with 100% PERV element inactivation.

Main Results:

• * Successfully inactivated all PERV elements in targeted porcine cells.
• * Demonstrated the efficacy of CRISPR-Cas9 in disrupting essential viral replication machinery.
• * Established a method for generating PERV-free porcine cells for xenotransplantation.

Conclusions:

• * CRISPR-Cas9 mediated PERV inactivation is a viable strategy for xenotransplantation safety.
• * This approach offers a potential solution to a major hurdle in utilizing porcine xenografts.
• * The described methodology supports the future application of PERV-free porcine cells in human transplantation.