Cancer Models in Xenopus tropicalis by CRISPR/Cas9 Mediated Knockout of Tumor Suppressors.

Abstract

The recent advent of CRISPR/Cas9 as a straightforward genome editing tool has allowed the establishment of the first bona fide genetic cancer models within the diploid aquatic model organism Xenopus tropicalis (X. tropicalis). Within this chapter, we demonstrate the methods for targeting tumor suppressors with the CRISPR/Cas9 system in the developing X. tropicalis embryo. We further illustrate genotyping and phenotyping of the resulting tumor-bearing F0 mosaic mutant animals (crispants). We focus in detail on the histopathological analysis of cancer neoplasms, the methodology to illustrate high proliferative index by proliferation marker immunofluorescence and how to isolate specific (tumor) cell populations by laser capture microdissection. As such, the described pipeline allows for rapid establishment of novel cancer models by CRISPR/Cas9 targeting of established tumor suppressor genes, or novel candidates obtained from clinical data. In conclusion, we thus provide the methodology for modeling human cancer with the highly efficient CRISPR/Cas9 system in F0 X. tropicalis.