Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.
Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.
Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.