An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination
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. These alleles permit numerous applications and have proven to be very useful. I...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-06-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/76077 |
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| author | Oguz Kanca Jonathan Zirin Yanhui Hu Burak Tepe Debdeep Dutta Wen-Wen Lin Liwen Ma Ming Ge Zhongyuan Zuo Lu-Ping Liu Robert W Levis Norbert Perrimon Hugo J Bellen |
| author_facet | Oguz Kanca Jonathan Zirin Yanhui Hu Burak Tepe Debdeep Dutta Wen-Wen Lin Liwen Ma Ming Ge Zhongyuan Zuo Lu-Ping Liu Robert W Levis Norbert Perrimon Hugo J Bellen |
| author_sort | Oguz Kanca |
| collection | DOAJ |
| description | 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. 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 (>5 kb). 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). 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. |
| first_indexed | 2024-04-12T16:38:24Z |
| format | Article |
| id | doaj.art-21349c0042e4440c9ed944c980a33c8b |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:38:24Z |
| publishDate | 2022-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-21349c0042e4440c9ed944c980a33c8b2022-12-22T03:24:53ZengeLife Sciences Publications LtdeLife2050-084X2022-06-011110.7554/eLife.76077An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombinationOguz Kanca0https://orcid.org/0000-0001-5438-0879Jonathan Zirin1Yanhui Hu2Burak Tepe3Debdeep Dutta4Wen-Wen Lin5Liwen Ma6Ming Ge7Zhongyuan Zuo8Lu-Ping Liu9Robert W Levis10https://orcid.org/0000-0003-3453-2390Norbert Perrimon11https://orcid.org/0000-0001-7542-472XHugo J Bellen12https://orcid.org/0000-0001-5992-5989Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Genetics, Harvard Medical School, Boston, United StatesDepartment of Genetics, Harvard Medical School, Boston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Duncan Neurological Research Institute, Texas Children Hospital, Houston, United StatesDepartment of Genetics, Harvard Medical School, Boston, United StatesDepartment of Embryology, Carnegie Institution for Science, Baltimore, United StatesDepartment of Genetics, Harvard Medical School, Boston, United States; Howard Hughes Medical Institute, Harvard Medical School, Boston, United StatesDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; Department of Neuroscience, Baylor College of Medicine, Houston, United StatesPreviously, 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. 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 (>5 kb). 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). 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.https://elifesciences.org/articles/76077CRISPRhomologous recombinationknock-inknock-outgene trapprotein trap |
| spellingShingle | Oguz Kanca Jonathan Zirin Yanhui Hu Burak Tepe Debdeep Dutta Wen-Wen Lin Liwen Ma Ming Ge Zhongyuan Zuo Lu-Ping Liu Robert W Levis Norbert Perrimon Hugo J Bellen An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination eLife CRISPR homologous recombination knock-in knock-out gene trap protein trap |
| title | An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination |
| title_full | An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination |
| title_fullStr | An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination |
| title_full_unstemmed | An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination |
| title_short | An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination |
| title_sort | expanded toolkit for drosophila gene tagging using synthesized homology donor constructs for crispr mediated homologous recombination |
| topic | CRISPR homologous recombination knock-in knock-out gene trap protein trap |
| url | https://elifesciences.org/articles/76077 |
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