Protocol for eVLP-mediated CRISPR-Cas9 and prime editing in pancreatic cancer cell lines
Abstract
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.
// Source
Authors: Chloe Timmins-Young, Marco Cordani, Elisa Giovannetti, Despoina Aslanoglou, Alvaro Gutierrez, Eithne Costello, Mark A. Glenn
Institutions: Vrije Universiteit Amsterdam, University of Liverpool, Amsterdam University Medical Centers, Universidad Complutense de Madrid, University of Portsmouth, Amsterdam UMC Location Vrije Universiteit Amsterdam, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Fondazione Pisa