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.