Rapid interrogation of cancer cell of origin through CRISPR editing

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.

Models to study the earliest stages in cancer progression must, by definition, start with normal cells to assess the consequences of a suspected oncogenic perturbation. Inbred mouse strains and genetically engineered mouse models (GEMMs) have, for decades, served as gold standards for such studies but require significant time (years) to generate, breed, and age mice. CRISPR technology has greatly accelerated the pace of generating GEMMs through delivery of sgRNAs (guide RNAs) and/or Cas9 to tissues such as lung and liver (1–3). The ability to grow primary tissues ex vivo as organoids and introduce precise genetic changes into these cultures provides an alternative platform to model genomic alterations with speed and efficiency, as reported for intestine and prostate (4–7). Here we demonstrate highly efficient (50 to 90%) editing of primary prostate epithelial organoid cultures, including multigenic or intrachromosomal (>2 Mb) deletions, through transient electroporation of Cas9–sgRNA ribonucleoprotein (cRNP) complexes. We also show cRNP-based CRISPR editing can be performed on freshly isolated prostate epithelial cells and then transplanted orthotopically into the prostates of recipient mice in a single day, enabling extremely rapid generation of in vivo cancer models in immunodeficient as well as immunocompetent settings. Finally, we show that this approach can be used to address cancer cell-of-origin questions by multigenic editing selectively in luminal versus basal epithelial cells.