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

Shivohum Bahuguna,Tianyu Wang,Michael Boutros,Jun Zhou,Fillip Port,Siamak Redhai

doi:10.3390/cells10113156

Shivohum Bahuguna, Tianyu Wang + Show 4 more

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https://doi.org/10.3390/cells10113156

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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.

Highlights

The application of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)—CRISPR-associated (Cas) systems for genomic studies has recently emerged as a powerful tool [1]
In the most widely used CRISPR system, the RNA-guided endonuclease Cas9 is targeted to specific DNA sequences to create double-strand breaks (DSBs) by a short guide-RNA sequence containing several nucleotides that are complementary to the target gene of interest [3,4]
In flies continuously expressing uM Cas9 with gRNA2x targeting neur and Notch, we observed a significant increase in intestinal progenitors such as stem cells (ISCs)/EB proliferation in both R2/R4 regions, the effect for Notch in the R2 region was more modest when compared to neur (Figures 3C,D and S1C)

Summary

Introduction

The application of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)—CRISPR-associated (Cas) systems for genomic studies has recently emerged as a powerful tool [1]. Discovered as a defense mechanism against invading viruses in bacteria and archaea, CRISPR-Cas has been adapted for genome engineering applications in many organisms [1,2]. In the most widely used CRISPR system, the RNA-guided endonuclease Cas is targeted to specific DNA sequences to create double-strand breaks (DSBs) by a short guide-RNA (gRNA) sequence containing several nucleotides that are complementary to the target gene of interest [3,4]. Cleavage of the target site and subsequent DSBs are repaired by the cellular DNA repair pathways, which includes the error prone non-homologous end joining pathway (NHEJ pathway) and the more precise homology-directed repair (HDR). In recent times, CRISPR-Cas has been used in various fields including cancer genetics [6]

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