CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato
Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a...
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MDPI AG
2020-12-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/12/1/59 |
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| author | Ilan Ben Shlush Aviva Samach Cathy Melamed-Bessudo Daniela Ben-Tov Tal Dahan-Meir Shdema Filler-Hayut Avraham A. Levy |
| author_facet | Ilan Ben Shlush Aviva Samach Cathy Melamed-Bessudo Daniela Ben-Tov Tal Dahan-Meir Shdema Filler-Hayut Avraham A. Levy |
| author_sort | Ilan Ben Shlush |
| collection | DOAJ |
| description | Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F<sub>1</sub> hybrids from divergent parental lines, each with a different mutation at the <i>Carotenoid isomerase</i> (<i>CRTISO</i>) locus. IHSR events, namely crossover or gene conversion (GC), between the two <i>CRTISO</i> mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F<sub>2</sub> seeds were grown to test for germinal transmission of HR events. Two out of five F<sub>1</sub> plants showing red sectors had their IHSR events germinally transmitted to F<sub>2</sub>, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools. |
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| last_indexed | 2024-03-10T13:34:31Z |
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| spelling | doaj.art-0ede88e0087340ba9b8e773b060d6b122023-11-21T07:38:39ZengMDPI AGGenes2073-44252020-12-011215910.3390/genes12010059CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in TomatoIlan Ben Shlush0Aviva Samach1Cathy Melamed-Bessudo2Daniela Ben-Tov3Tal Dahan-Meir4Shdema Filler-Hayut5Avraham A. Levy6Department of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelDepartment of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, IsraelHomologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F<sub>1</sub> hybrids from divergent parental lines, each with a different mutation at the <i>Carotenoid isomerase</i> (<i>CRTISO</i>) locus. IHSR events, namely crossover or gene conversion (GC), between the two <i>CRTISO</i> mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F<sub>2</sub> seeds were grown to test for germinal transmission of HR events. Two out of five F<sub>1</sub> plants showing red sectors had their IHSR events germinally transmitted to F<sub>2</sub>, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.https://www.mdpi.com/2073-4425/12/1/59DNA double-strand break repairhomologous recombinationCRISPR/Cas9inter homologous somatic recombinationtargeted gene conversiontargeted crossover |
| spellingShingle | Ilan Ben Shlush Aviva Samach Cathy Melamed-Bessudo Daniela Ben-Tov Tal Dahan-Meir Shdema Filler-Hayut Avraham A. Levy CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato Genes DNA double-strand break repair homologous recombination CRISPR/Cas9 inter homologous somatic recombination targeted gene conversion targeted crossover |
| title | CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato |
| title_full | CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato |
| title_fullStr | CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato |
| title_full_unstemmed | CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato |
| title_short | CRISPR/Cas9 Induced Somatic Recombination at the <i>CRTISO</i> Locus in Tomato |
| title_sort | crispr cas9 induced somatic recombination at the i crtiso i locus in tomato |
| topic | DNA double-strand break repair homologous recombination CRISPR/Cas9 inter homologous somatic recombination targeted gene conversion targeted crossover |
| url | https://www.mdpi.com/2073-4425/12/1/59 |
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