Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors
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 <i>Drosophila melanogaster</i> to assess its ability to induce stem cell-derived tumo...
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MDPI AG
2021-11-01
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| author | Shivohum Bahuguna Siamak Redhai Jun Zhou Tianyu Wang Fillip Port Michael Boutros |
| author_facet | Shivohum Bahuguna Siamak Redhai Jun Zhou Tianyu Wang Fillip Port Michael Boutros |
| author_sort | Shivohum Bahuguna |
| collection | DOAJ |
| description | 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 <i>Drosophila melanogaster</i> 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, <i>Drosophila</i> can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo. |
| first_indexed | 2024-03-10T05:35:58Z |
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| id | doaj.art-06e7ab93f39740eaa54894aa8ff5c1ce |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T05:35:58Z |
| publishDate | 2021-11-01 |
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| series | Cells |
| spelling | doaj.art-06e7ab93f39740eaa54894aa8ff5c1ce2023-11-22T22:52:06ZengMDPI AGCells2073-44092021-11-011011315610.3390/cells10113156Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal TumorsShivohum Bahuguna0Siamak Redhai1Jun Zhou2Tianyu Wang3Fillip Port4Michael Boutros5German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyGerman Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyGerman Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyGerman Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyGerman Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyGerman Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, BioQuant and Medical Faculty Mannheim, Heidelberg University, D-69120 Heidelberg, GermanyCRISPR-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 <i>Drosophila melanogaster</i> 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, <i>Drosophila</i> can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.https://www.mdpi.com/2073-4409/10/11/3156CRISPRCas9tumorsBMPNotchJNK |
| spellingShingle | Shivohum Bahuguna Siamak Redhai Jun Zhou Tianyu Wang Fillip Port Michael Boutros Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors Cells CRISPR Cas9 tumors BMP Notch JNK |
| title | Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors |
| title_full | Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors |
| title_fullStr | Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors |
| title_full_unstemmed | Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors |
| title_short | Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors |
| title_sort | conditional crispr cas genome editing in i drosophila i to generate intestinal tumors |
| topic | CRISPR Cas9 tumors BMP Notch JNK |
| url | https://www.mdpi.com/2073-4409/10/11/3156 |
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