Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV

Hongzhen Mao
,
Jinyang Li
,
Mengyu Gao
ORCID logo ,
Xinmei Liu
,
Haohan Zhang
,
Yijia Zhuang
,
Tianyi He
,
Wei Zuo
,
Lang Bai
,
Ji Bao
* ORCID logo
Version 1 : Received: 25 March 2024 / Approved: 25 March 2024 / Online: 26 March 2024 (13:06:12 CET)

A peer-reviewed article of this Preprint also exists.

Mao, H.; Li, J.; Gao, M.; Liu, X.; Zhang, H.; Zhuang, Y.; He, T.; Zuo, W.; Bai, L.; Bao, J. Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV. Microorganisms 2024, 12, 837. Mao, H.; Li, J.; Gao, M.; Liu, X.; Zhang, H.; Zhuang, Y.; He, T.; Zuo, W.; Bai, L.; Bao, J. Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV. Microorganisms 2024, 12, 837.

Abstract

In the world's first pig-to-human cardiac xenotransplant, the elevated levels of porcine cytomeg-alovirus (PCMV) are considered key factors contributing to patient mortality. This has renewed attention to PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effec-tively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infec-tion capabilities. This opens up possibilities for the future production of pig populations with anti-viral functionalities.

Keywords

porcine cytomegalovirus; CRISPR/Cas9; RNAi; knock-in; site-specific integration

Subject

Biology and Life Sciences, Virology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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