Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors

Abstract

CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.

Restraining Wnt activation and intestinal tumorigenesis by a Rab35 dependent GTPase relay

“…For experiments where temperature shift was not required, flies were kept at 29°C to ensure age-dependent factors do not confound comparison between experiments. For CRISPR mutagenesis experiments, newly eclosed flies were shifted to 29°C for 10 days and subsequently to 18°C for 30 days before switching back to 29°C for one day prior to imaging as previously reported [91]. For all experiments, mated female flies were used and transferred to fresh food every two days.…”

Molecular regulation after mucosal injury and regeneration in ulcerative colitis

“…However, due to early death of the mice, the effect of Hes1 deficiency on adult mice is uncertain ( Tsai et al, 2022 ). The Notch pathway negatively regulates the differentiation of secretory cells after the inhibition of Math1, a target gene in the Notch pathway, promoting RBP-J expression ( Bahuguna et al, 2021 ). In addition, loss of Math1 causes the abnormal differentiation of mucosal goblet cells, leading to loss of the colonic mucus layer, which induces UC ( Shi et al, 2021 ).…”