Rapid interrogation of cancer cell of origin through CRISPR editing

Significance Modeling cancer formation requires introduction of relevant oncogenic perturbations into normal cells in a tissue/cell-type–specific manner. Genetically engineered mouse models are powerful but require significant time and cost to generate and maintain. The ability to edit primary epithelial cells ex vivo followed by orthotopic transplantation provides an alternative strategy for cancer modeling but requires efficient gene editing, typically in a multiplex fashion. Here we successfully engineer multigenic perturbations or chromosomal rearrangements in primary prostate organoids through single-step Cas9–sgRNA ribonucleoprotein electroporation. This approach can also address cell-of-origin questions by directly editing and transplanting freshly isolated subpopulations without the intermediate step of organoid culture, providing a rapid complement to traditional lineage tracing approaches. The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9–sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.