Engineering the Drosophila Genome for Developmental Biology

Korona, Dagmara; Koestler, Stefan A.; Russell, Steven

doi:10.3390/jdb5040016

Cited by 20 publications

(20 citation statements)

References 144 publications

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“…Scarless gene editing can be achieved through the use of single-stranded DNA donors ( Gratz et al, 2014 ; Xue et al, 2014 ). However, they are limited in size to ~200 nucleotides by current synthesis methods ( Korona et al, 2017 ). Accordingly, sgRNA sites must be close to the specific nucleotides to be edited and because they cannot carry visible markers they require laborious screening methods to find flies carrying the correct gene editing event.…”

Section: Discussionmentioning

confidence: 99%

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Li‐Kroeger

Kanca

Lee

et al. 2018

eLife

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We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

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Section: Discussionmentioning

confidence: 99%

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Li‐Kroeger

Kanca

Lee

et al. 2018

eLife

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“…Cloning gRNAs. To generate the transgenic fly lines carrying the tagged isoforms, we used CRISPR/Cas9 technology as previously described [37]. We initially designed the insertion sites as indicated (Figure 1) and choose appropriate gRNAs (Table S3) that were cloned into pCDF3 or pCDF4 [57].…”

Section: Methodsmentioning

confidence: 99%

“…In order to determine the expression and localisation of specific Sgg proteoforms we used CRISPR/Cas9 genome engineering to introduce different in-frame protein tags into specific exons at the endogenous sgg locus [37]. We first focused on the major C terminal proteoforms represented by Sgg-PA and Sgg-PB, constructing fly lines containing a 3xFLAG-StrepTagII-mVenus-StrepTagII (FSVS) cassette just before the termination codon.…”

Section: In Vivo Tagging Of Major Sgg Proteoformsmentioning

confidence: 99%

“…To help address the role of alternative Sgg proteoforms in Drosophila and the developmental roles GSK-3 plays, as well as contributing to the debate surrounding the functionality of splice isoforms, we used a CRISPR-Cas9 based genome engineering strategy to tag specific Sgg proteoforms. We introduced fluorescent protein or affinity tags into the endogenous sgg locus in Drosophila [37], altogether tagging eight different exons. These tagged Sgg proteoforms allowed us to follow their expression across embryonic development by immunohistochemistry and/or fluorescence microscopy, revealing unique and specific expression for each of the tagged proteoforms.…”

Section: Introductionmentioning

confidence: 99%

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Characterisation of protein isoforms encoded by theDrosophilaGlycogen Synthase Kinase 3 geneshaggy

Korona

Nightingale

Fabre

et al. 2019

Preprint

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The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoform classes we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β encoded by independent vertebrate genes. Our analysis suggests that different protein isoforms generated by alternative splicing perform distinct functions.

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“…They are inexpensive to grow, have a short life cycle, and have well-developed genetic tools for probing biological pathways (Griffin, Binari, and Perrimon 2014;Venken and Bellen 2014;Korona, Koestler, and Russell 2017). These features coupled with the evolutionary conservation of biological processes found in higher organisms like humans have made flies a prime model organism (Adams et al 2000;Wangler et al 2017).…”

Section: Introductionmentioning

confidence: 99%

The High-throughput WAFFL System for Treating and Monitoring Individual Drosophila melanogaster Adults

Jaime

Karrot

Salem

et al. 2018

Preprint

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Non-mammalian model organisms have been essential for our understanding of the mechanisms and control of development, disease, and physiology, but are underutilized in pharmacological phenotypic screening assays due to low throughput compared to cell-based systems. To increase the utility of using Drosophila melanogaster in screening, we have designed the whole animal feeding flat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high throughput format. Our system was conceived keeping in mind the use of off-the-shelf, commercial, 96-well consumables and equipment in order to be amenable to experimental needs.Here we provide an overview of the design and 3-D printing manufacture specifications. J a i m e e t a l .

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Engineering the Drosophila Genome for Developmental Biology

Cited by 20 publications

References 144 publications

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Characterisation of protein isoforms encoded by theDrosophilaGlycogen Synthase Kinase 3 geneshaggy

The High-throughput WAFFL System for Treating and Monitoring Individual Drosophila melanogaster Adults

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