STAR protocolsJul 7, 2026

Method for creating pig fetal cells with multiple precise genetic edits

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 8, 2026

Abstract

Multiplexed prime editing has been successfully demonstrated in porcine fetal fibroblasts (PFFs) with the introduction of three Alzheimer's disease-associated mutations.

Porcine fetal fibroblasts serve as standard donor cells for creating cloned pigs.
Prime editing allows for precise modifications to the genome.
A detailed protocol for generating monoclonal cell lines via multiplexed prime editing is described.
The method can be adapted for editing other genetic loci in porcine cells.

Simplified

Porcine fetal fibroblasts (PFFs) serve as standard donor cells for generating cloned pigs, and prime editing (PE) enables precise genome modification. Here, we describe a protocol for generating multiplexed prime-edited monoclonal cell lines in PFFs. We describe steps for pegRNA/ngRNA design and screening, plasmid electroporation, nocodazole treatment, puromycin selection, and monoclonal isolation and genotyping. Although demonstrated by introducing three Alzheimer's disease-associated pathogenic mutations, the pipeline can be readily adapted to multiplex PE of other endogenous loci in porcine cells. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

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Funding

Competing interests

Declaration of interests The authors have filed a patent application related to the findings of this study.

PubMed

Method for creating pig fetal cells with multiple precise genetic edits

Abstract

Full Text

Funding

Competing interests

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