An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila
- Baylor College of Medicine, United States
- Rice University, United States
Abstract
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.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. The whole genome sequencing files are available on Zenodo (https://zenodo.org/record/1341241).
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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila - sequence filesPublicly available on Zenodo under a Creative Commons Attribution 4.0 License.
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Author details
Funding
National Institutes of Health (R01GM067858)
- Hugo J Bellen
Howard Hughes Medical Institute
- Hugo J Bellen
Robert A. and Renee E. Belfer Family Foundation
- Hugo J Bellen
Huffington Foundation
- Hugo J Bellen
National Institutes of Health (Training grant T32NS043124)
- David Li-Kroeger
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Li-Kroeger et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila
eLife 7:e38709.
https://doi.org/10.7554/eLife.38709
