Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9
Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene...
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| Published in | Nature communications Vol. 11; no. 1; pp. 1178 - 10 |
|---|---|
| Main Authors | , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
04.03.2020
Nature Publishing Group Nature Portfolio |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2041-1723 2041-1723 |
| DOI | 10.1038/s41467-020-14981-y |
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| Abstract | Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.
Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content. |
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| AbstractList | Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops. Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops. Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops. Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content. |
| ArticleNumber | 1178 |
| Author | Lipzen, Anna Schmutz, Jeremy Dong, Oliver Xiaoou Barry, Kerrie W. Ronald, Pamela C. Duong, Phat Q. Zhang, Nan Tian, Li Butler, Corinne Jain, Rashmi Martin, Joel A. Li, Yan Yu, Shu |
| Author_xml | – sequence: 1 givenname: Oliver Xiaoou orcidid: 0000-0003-4692-8659 surname: Dong fullname: Dong, Oliver Xiaoou organization: Department of Plant Pathology and the Genome Center, University of California, Innovative Genomics Institute, Feedstocks Division, The Joint Bioenergy Institute – sequence: 2 givenname: Shu surname: Yu fullname: Yu, Shu organization: Department of Plant Sciences, University of California – sequence: 3 givenname: Rashmi orcidid: 0000-0001-6819-847X surname: Jain fullname: Jain, Rashmi organization: Department of Plant Pathology and the Genome Center, University of California – sequence: 4 givenname: Nan surname: Zhang fullname: Zhang, Nan organization: Department of Plant Pathology and the Genome Center, University of California – sequence: 5 givenname: Phat Q. surname: Duong fullname: Duong, Phat Q. organization: Department of Plant Pathology and the Genome Center, University of California – sequence: 6 givenname: Corinne surname: Butler fullname: Butler, Corinne organization: Department of Plant Pathology and the Genome Center, University of California – sequence: 7 givenname: Yan surname: Li fullname: Li, Yan organization: Department of Plant Pathology and the Genome Center, University of California – sequence: 8 givenname: Anna surname: Lipzen fullname: Lipzen, Anna organization: Department of Energy Joint Genome Institute – sequence: 9 givenname: Joel A. orcidid: 0000-0001-9511-6441 surname: Martin fullname: Martin, Joel A. organization: Department of Energy Joint Genome Institute – sequence: 10 givenname: Kerrie W. surname: Barry fullname: Barry, Kerrie W. organization: Department of Energy Joint Genome Institute – sequence: 11 givenname: Jeremy orcidid: 0000-0001-8062-9172 surname: Schmutz fullname: Schmutz, Jeremy organization: Department of Energy Joint Genome Institute – sequence: 12 givenname: Li orcidid: 0000-0001-6461-6072 surname: Tian fullname: Tian, Li organization: Department of Plant Sciences, University of California – sequence: 13 givenname: Pamela C. orcidid: 0000-0002-4107-1345 surname: Ronald fullname: Ronald, Pamela C. email: pcronald@ucdavis.edu organization: Department of Plant Pathology and the Genome Center, University of California, Innovative Genomics Institute, Feedstocks Division, The Joint Bioenergy Institute |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/32132530$$D View this record in MEDLINE/PubMed https://www.osti.gov/servlets/purl/1615316$$D View this record in Osti.gov |
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| ContentType | Journal Article |
| Copyright | The Author(s) 2020 This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. |
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| CorporateAuthor | Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States) |
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| Snippet | Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through... Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through... Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an... |
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| SubjectTerms | 13/106 38/22 38/23 38/77 45/29 631/449/1659 631/449/447/2311 631/449/711 631/61/318 BASIC BIOLOGICAL SCIENCES Biosynthesis Biosynthetic Pathways - genetics Carotenoids Carotenoids - analysis Carotenoids - metabolism Cereal crops CRISPR CRISPR-Cas Systems - genetics Deoxyribonucleic acid DNA DNA, Plant - genetics Gene Editing - methods Gene Knock-In Techniques - methods Gene sequencing Genome editing Genome, Plant - genetics Genomes Genomics Humanities and Social Sciences Insertion Inserts Markers Morphology multidisciplinary Mutation Oryza - genetics Oryza - metabolism Plant Breeding - methods Plants, Genetically Modified Rice Science Science (multidisciplinary) Seeds Seeds - chemistry Transgenes Transgenic plants Whole Genome Sequencing |
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| Title | Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9 |
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