One-step CRISPR-Cas9 protocol for the generation of plug &amp; play conditional knockouts in Drosophila melanogaster

Yu, Joyce J.S.; Vincent, Jean‐Paul; McGough, Ian J.

doi:10.1016/j.xpro.2021.100560

Cited by 2 publications

(2 citation statements)

References 11 publications

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“…This is especially important when testing, for example, mutant transgenes in follow up experiments. Note: Detailed CRISPR protocols have been described ( Hoppe and Ashe, 2021a ; Yu et al., 2021 ), including methods for scarless editing ( Lamb et al., 2017 ; Nyberg et al., 2020 ). …”

Section: Before You Beginmentioning

confidence: 99%

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Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

2021

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Summary Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021) .

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Section: Before You Beginmentioning

confidence: 99%

“…Note: Detailed CRISPR protocols have been described ( Hoppe and Ashe, 2021a ; Yu et al., 2021 ), including methods for scarless editing ( Lamb et al., 2017 ; Nyberg et al., 2020 ).…”

Section: Before You Beginmentioning

confidence: 99%

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

2021

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A simple MiMIC-based approach for tagging endogenous genes to visualise live transcription in Drosophila

Beadle,

Sutcliffe,

Ashe

2024

Development

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Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem loops into endogenous genes requires laborious CRISPR genome editing. Here we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging and live imaging transcription of endogenous genes and the long non-coding RNA, roX1, in the embryo. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.

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One-step CRISPR-Cas9 protocol for the generation of plug & play conditional knockouts in Drosophila melanogaster

Cited by 2 publications

References 11 publications

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

A simple MiMIC-based approach for tagging endogenous genes to visualise live transcription in Drosophila

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