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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Autores principales:	Shivohum Bahuguna, Siamak Redhai, Jun Zhou, Tianyu Wang, Fillip Port, Michael Boutros
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	CRISPR Cas9 tumors BMP Notch JNK Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/06e7ab93f39740eaa54894aa8ff5c1ce
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spelling	oai:doaj.org-article:06e7ab93f39740eaa54894aa8ff5c1ce2021-11-25T17:12:05ZConditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors10.3390/cells101131562073-4409https://doaj.org/article/06e7ab93f39740eaa54894aa8ff5c1ce2021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4409/10/11/3156https://doaj.org/toc/2073-4409CRISPR-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.Shivohum BahugunaSiamak RedhaiJun ZhouTianyu WangFillip PortMichael BoutrosMDPI AGarticleCRISPRCas9tumorsBMPNotchJNKBiology (General)QH301-705.5ENCells, Vol 10, Iss 3156, p 3156 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Cas9 tumors BMP Notch JNK Biology (General) QH301-705.5
spellingShingle	CRISPR Cas9 tumors BMP Notch JNK Biology (General) QH301-705.5 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
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.
format	article
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
title	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_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_sort	conditional crispr-cas genome editing in <i>drosophila</i> to generate intestinal tumors
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/06e7ab93f39740eaa54894aa8ff5c1ce
work_keys_str_mv	AT shivohumbahuguna conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors AT siamakredhai conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors AT junzhou conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors AT tianyuwang conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors AT fillipport conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors AT michaelboutros conditionalcrisprcasgenomeeditinginidrosophilaitogenerateintestinaltumors
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Conditional CRISPR-Cas Genome Editing in <i>Drosophila</i> to Generate Intestinal Tumors

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