Protocol for eVLP-mediated CRISPR-Cas9 and prime editing in pancreatic cancer cell lines
Pancreatic ductal adenocarcinoma (PDAC) cell lines resist transfection, limiting gene-editing experiments. Here, we present a protocol for delivering CRISPR-Cas9 or prime editors into PDAC cell lines using retrovirus-derived engineered virus-like particles (eVLPs). We describe steps for generating eVLPs, packaging CRISPR-Cas9 ribonucleoproteins or prime editors with engineered prime-editing guide RNAs (epegRNAs), and transducing human PDAC cells. We use the HEK3 locus and NQO1 (NAD(P)H quinone dehydrogenase 1) exon 6 as example edits and controls, respectively. This protocol adapts readily to other genomic sites.
Authors
- Eithne Costello (ORCID: https://orcid.org/0000-0002-0104-8992)
- Elisa Giovannetti (ORCID: https://orcid.org/0000-0002-7565-7504)
- Mark A. Glenn (ORCID: https://orcid.org/0009-0005-2490-2740)
- Despoina Aslanoglou (ORCID: https://orcid.org/0000-0002-8444-1385)
- Marco Cordani (ORCID: https://orcid.org/0000-0001-9342-4862)
- Alvaro Gutierrez
- Chloe Timmins-Young (ORCID: https://orcid.org/0009-0006-5663-9120)
Institutions
- Universidad Complutense de Madrid (ES)
- University of Liverpool (GB)
- Fondazione Pisa (IT)
- Hospital Clínico San Carlos (ES)
- Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (ES)
- Amsterdam University Medical Centers (NL)
- University of Portsmouth (GB)
- Vrije Universiteit Amsterdam (NL)
- Amsterdam UMC Location Vrije Universiteit Amsterdam (NL)
Publication Details
- Journal
- STAR Protocols
- Published
- 2026-10-17
- DOI
- https://doi.org/10.1016/j.xpro.2026.104904
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Cancer Research UK
- European Cooperation in Science and Technology
- KWF Kankerbestrijding
- Associazione Italiana per la Ricerca sul Cancro