A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila
Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2020-10-01
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| Series: | Fly |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/19336934.2020.1832416 |
| _version_ | 1797678370399453184 |
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| author | Kevin G. Nyberg Joseph Q. Nguyen Yong-Jae Kwon Shelby Blythe Greg J. Beitel Richard Carthew |
| author_facet | Kevin G. Nyberg Joseph Q. Nguyen Yong-Jae Kwon Shelby Blythe Greg J. Beitel Richard Carthew |
| author_sort | Kevin G. Nyberg |
| collection | DOAJ |
| description | Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes. |
| first_indexed | 2024-03-11T22:58:44Z |
| format | Article |
| id | doaj.art-8a34f8df85884985b1caa1ddcad3a880 |
| institution | Directory Open Access Journal |
| issn | 1933-6934 1933-6942 |
| language | English |
| last_indexed | 2024-03-11T22:58:44Z |
| publishDate | 2020-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Fly |
| spelling | doaj.art-8a34f8df85884985b1caa1ddcad3a8802023-09-21T15:09:08ZengTaylor & Francis GroupFly1933-69341933-69422020-10-01141-4344810.1080/19336934.2020.18324161832416A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in DrosophilaKevin G. Nyberg0Joseph Q. Nguyen1Yong-Jae Kwon2Shelby Blythe3Greg J. Beitel4Richard Carthew5Northwestern UniversityNorthwestern UniversityNorthwestern UniversityNorthwestern UniversityNorthwestern UniversityNorthwestern UniversityGenome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.http://dx.doi.org/10.1080/19336934.2020.1832416crisprgene editingdrosophilahomology dependent repair |
| spellingShingle | Kevin G. Nyberg Joseph Q. Nguyen Yong-Jae Kwon Shelby Blythe Greg J. Beitel Richard Carthew A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila Fly crispr gene editing drosophila homology dependent repair |
| title | A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila |
| title_full | A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila |
| title_fullStr | A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila |
| title_full_unstemmed | A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila |
| title_short | A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila |
| title_sort | pipeline for precise and efficient genome editing by sgrna cas9 rnps in drosophila |
| topic | crispr gene editing drosophila homology dependent repair |
| url | http://dx.doi.org/10.1080/19336934.2020.1832416 |
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