An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination
- Baylor College of Medicine, United States
- Harvard Medical School, United States
- Carnegie Institution for Science, United States
Abstract
Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.
Data availability
Source data for the graphs in Supplementary table 1 is included above the graphs in Supplementary table 1. The list of all the generated alleles and the method used can be found in Supplementary table 2.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R01GM067858)
- Hugo J Bellen
Office of Research Infrastructure Programs, National Institutes of Health (R24OD031447)
- Oguz Kanca
- Hugo J Bellen
Office of Research Infrastructure Programs, National Institutes of Health (R24OD031447)
- Hugo J Bellen
National Institute of Neurological Disorders and Stroke (U54NS093793)
- Hugo J Bellen
Huffington Foundation
- Hugo J Bellen
National Institute of General Medical Sciences (GM067761)
- Jonathan Zirin
- Yanhui Hu
- Norbert Perrimon
National Institute of General Medical Sciences (GM084947)
- Jonathan Zirin
- Yanhui Hu
- Norbert Perrimon
Howard Hughes Medical Institute
- Norbert Perrimon
Carnegie Institution for Science
- Robert W Levis
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
Version history
- Received: December 7, 2021
- Preprint posted: December 24, 2021 (view preprint)
- Accepted: June 19, 2022
- Accepted Manuscript published: June 20, 2022 (version 1)
- Version of Record published: June 28, 2022 (version 2)
Copyright
© 2022, Kanca 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 Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination
eLife 11:e76077.
https://doi.org/10.7554/eLife.76077
Further reading
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- Chromosomes and Gene Expression
We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.
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- Genetics and Genomics
Telomeres, which are chromosomal end structures, play a crucial role in maintaining genome stability and integrity in eukaryotes. In the baker’s yeast Saccharomyces cerevisiae, the X- and Y’-elements are subtelomeric repetitive sequences found in all 32 and 17 telomeres, respectively. While the Y’-elements serve as a backup for telomere functions in cells lacking telomerase, the function of the X-elements remains unclear. This study utilized the S. cerevisiae strain SY12, which has three chromosomes and six telomeres, to investigate the role of X-elements (as well as Y’-elements) in telomere maintenance. Deletion of Y’-elements (SY12YΔ), X-elements (SY12XYΔ+Y), or both X- and Y’-elements (SY12XYΔ) did not impact the length of the terminal TG1-3 tracks or telomere silencing. However, inactivation of telomerase in SY12YΔ, SY12XYΔ+Y, and SY12XYΔ cells resulted in cellular senescence and the generation of survivors. These survivors either maintained their telomeres through homologous recombination-dependent TG1-3 track elongation or underwent microhomology-mediated intra-chromosomal end-to-end joining. Our findings indicate the non-essential role of subtelomeric X- and Y’-elements in telomere regulation in both telomerase-proficient and telomerase-null cells and suggest that these elements may represent remnants of S. cerevisiae genome evolution. Furthermore, strains with fewer or no subtelomeric elements exhibit more concise telomere structures and offer potential models for future studies in telomere biology.
