Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9
Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2017-01-01
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| Series: | Fly |
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| Online Access: | http://dx.doi.org/10.1080/19336934.2016.1220463 |
| _version_ | 1797678357292253184 |
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| author | Abigail M. Lamb Elizabeth A. Walker Patricia J. Wittkopp |
| author_facet | Abigail M. Lamb Elizabeth A. Walker Patricia J. Wittkopp |
| author_sort | Abigail M. Lamb |
| collection | DOAJ |
| description | Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as “scars.” To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, “scarless” edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species. |
| first_indexed | 2024-03-11T22:58:33Z |
| format | Article |
| id | doaj.art-5d09bdb8ef674aa59021343ba1098187 |
| institution | Directory Open Access Journal |
| issn | 1933-6934 1933-6942 |
| language | English |
| last_indexed | 2024-03-11T22:58:33Z |
| publishDate | 2017-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Fly |
| spelling | doaj.art-5d09bdb8ef674aa59021343ba10981872023-09-21T15:09:08ZengTaylor & Francis GroupFly1933-69341933-69422017-01-01111536410.1080/19336934.2016.12204631220463Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9Abigail M. Lamb0Elizabeth A. Walker1Patricia J. Wittkopp2University of MichiganUniversity of MichiganUniversity of MichiganGenome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as “scars.” To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, “scarless” edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.http://dx.doi.org/10.1080/19336934.2016.1220463crisprgenome editinghomology directed repairnon-model speciespoint mutationsingle-nucleotidetransgenic |
| spellingShingle | Abigail M. Lamb Elizabeth A. Walker Patricia J. Wittkopp Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 Fly crispr genome editing homology directed repair non-model species point mutation single-nucleotide transgenic |
| title | Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 |
| title_full | Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 |
| title_fullStr | Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 |
| title_full_unstemmed | Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 |
| title_short | Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9 |
| title_sort | tools and strategies for scarless allele replacement in drosophila using crispr cas9 |
| topic | crispr genome editing homology directed repair non-model species point mutation single-nucleotide transgenic |
| url | http://dx.doi.org/10.1080/19336934.2016.1220463 |
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