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      The vestigial Quadrant Enhancer is dispensable for pattern formation and development of the Drosophila wing

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      Author(s): 1 , 1 , 1 , 2 , 1 , §
      Other contributor(s): (Reviewer)
      Publication date (Electronic, pub):
      Publication date (Electronic, collection):
      Journal: microPublication Biology
      Publisher: Caltech Library

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          Abstract

          In Drosophila , the pattern of the wing selector gene, vestigial ( vg ), is established by at least two enhancers: the Boundary Enhancer, which drives expression along the disc’s Dorsal-Ventral boundary; and the Quadrant Enhancer (QE) that patterns the rest of the wing pouch. Using CRISPR/Cas9 editing, we deleted DNA fragments around the reported QE sequence and found that the full Vg pattern is formed. Furthermore, adult wings arising from these gene-edited animals are normal in shape and pattern, but slightly smaller in size, although this reduction is not wing-specific in males. We suggest that other enhancers act redundantly to establish the vg pattern and rescue wing development.

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          Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

          Fillip Port, Hui-Min Chen, Tzumin Lee … (2014)
          The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
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            Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

            Scott Gratz, Fiona Ukken, C Dustin Rubinstein … (2014)
            We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.
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              The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP.

              Duojia Pan, José M. Barrera, José Jiun-Shian Wu … (2005)
              Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Here we identify Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene.
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                Author and article information

                Journal
                Journal ID (nlm-ta): MicroPubl Biol
                Journal ID (iso-abbrev): MicroPubl Biol
                Title: microPublication Biology
                Publisher: Caltech Library
                ISSN (Electronic): 2578-9430
                Publication date (Electronic, pub): 13 June 2022
                Publication date (Electronic, collection): 2022
                Volume: 2022
                Electronic Location Identifier: 10.17912/micropub.biology.000585
                Affiliations
                [1 ] Department of Physiology, Biophysics, and Neurosciences, Centre for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN)
                [2 ] Department of Biological Sciences, University of Arkansas
                Author notes
                [§ ] Correspondence to: Marcos Nahmad ( mnahmad@ 123456fisio.cinvestav.mx )

                KF: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Visualization

                TM: Conceptualization, Methodology

                RR: Data curation, Formal analysis

                TE: Conceptualization, Formal analysis, Methodology, Resources, Supervision, Validation, Writing - original draft, Visualization

                MN: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing

                Article
                DOI: 10.17912/micropub.biology.000585
                PMC ID: 9242444
                SO-VID: 72e6e9a7-0f81-4cb5-9910-c7b2206f9940
                Copyright © Copyright: © 2022 by the authors
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 2 April 2022
                Date revision received : 7 June 2022
                Date accepted : 9 June 2022
                Categories
                Subject: New Finding
                Subject: Genotype Data
                Subject: Drosophila

                Data availability:

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