A CRISPR/Cas9-mediated Screening of the F-box domain-containing E3 Ligase family in Intestinal Organoids


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Hossein Kashfi1,Nicholas Jinks1,Abdolrahman Shams Nateri1
1Cancer Genetic and Stem cell group, Division of cancer and stem cell, Nottingham University

Abstract

Background

Organoid culture faithfully reproduces in vitro the in vivo structure of the intestinal/colon epithelium and allows elucidating molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumourigenesis.

Now, in the present study, we applied the CRISPR/Cas9-mediated engineering method for screening of 34 F-box domain-containing E3-ligases in intestinal organoids. As the biological activity of the majority of F-box proteins (FBPs) in colorectal cancer (CRC) is elusive, exploring the function of FBPs family would further provide promising information on their role in CRC tumourigenesis

Method

The gRNA library of F-box proteins was validated through sequencing, restriction enzyme digestion and generation of stable cell lines in cultured cells. The lentivirus containing CRISPR gRNA were generated for further transduction of intestinal organoids. Furthermore, the transgenic organoids line expressing cas9 were generated through lentiviral transduction.

Following transduction of cas9 organoids, the phenotypical/morphological alterations of the K/O organoids were evaluated for selecting the candidates and further downstream analysis.

Results

Among 34 K/O organoids for F-box proteins, the K/O FBXL18 and K/O FBXL17 showed significant morphological changes in comparison to their controls. The K/O FBXL18 organoids failed to differentiate and, during the following organogenesis, failed to expand and mature like control counterparts. Suppression of proliferation and increase in apoptosis were also detected in K/O FBXL17 organoids. Downstream analysis of the K/O FBXL18 in wound healing scratch assay showed a significantly decreased movement of cells migration in comparison to control Cas9 DLD-1group (Pā€‰<ā€‰0.01, respectively). Additionally, the colony formation assay also showed a significant decrease in the number of colonies compared with the Cas9 DLD-1 control group (Pā€‰<ā€‰0.01).

Conclusion

FBPs K/O intestinal organoids led to phenotypical alterations that could be associated with proliferation and differentiation. CRISPR/Cas9 organoid mutagenesis can be utilised for screening of candidate genes for possible involvement in tumorigenesis.