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Title: Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

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.

Authors:
ORCiD logo [1];  [2]; ORCiD logo [2];  [2];  [2];  [2];  [2];  [3]; ORCiD logo [3];  [3]; ORCiD logo [3]; ORCiD logo [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Univ. of California, Davis, CA (United States); Innovative Genomics Inst., Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  2. Univ. of California, Davis, CA (United States)
  3. USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1615316
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Dong, Oliver Xiaoou, Yu, Shu, Jain, Rashmi, Zhang, Nan, Duong, Phat Q., Butler, Corinne, Li, Yan, Lipzen, Anna, Martin, Joel A., Barry, Kerrie W., Schmutz, Jeremy, Tian, Li, and Ronald, Pamela C. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. United States: N. p., 2020. Web. doi:10.1038/s41467-020-14981-y.
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Dong, Oliver Xiaoou, Yu, Shu, Jain, Rashmi, Zhang, Nan, Duong, Phat Q., Butler, Corinne, Li, Yan, Lipzen, Anna, Martin, Joel A., Barry, Kerrie W., Schmutz, Jeremy, Tian, Li, & Ronald, Pamela C. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. United States. https://doi.org/10.1038/s41467-020-14981-y
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Dong, Oliver Xiaoou, Yu, Shu, Jain, Rashmi, Zhang, Nan, Duong, Phat Q., Butler, Corinne, Li, Yan, Lipzen, Anna, Martin, Joel A., Barry, Kerrie W., Schmutz, Jeremy, Tian, Li, and Ronald, Pamela C. Wed . "Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9". United States. https://doi.org/10.1038/s41467-020-14981-y. https://www.osti.gov/servlets/purl/1615316.
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@article{osti_1615316,
title = {Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9},
author = {Dong, Oliver Xiaoou and Yu, Shu and Jain, Rashmi and Zhang, Nan and Duong, Phat Q. and Butler, Corinne and Li, Yan and Lipzen, Anna and Martin, Joel A. and Barry, Kerrie W. and Schmutz, Jeremy and Tian, Li and Ronald, Pamela C.},
abstractNote = {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.},
doi = {10.1038/s41467-020-14981-y},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {2020},
month = {3}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1038/s41467-020-14981-y
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Figures / Tables:

Fig. 1 Fig. 1: Scheme for targeted insertion of the carotenoid cassette. a Map of the donor plasmid pAcc-B with details of the carotenoid cassette (orange arrow). Red and blue arrows represent the homology arms. The two vertical green triangles mark the positions of the guide RNA B target sites. Themore » nucleotide sequence of the donor plasmid is presented in Supplementary Data 1. Primers used to genotype the donor plasmid are marked on the map. b Map of the CRISPR plasmid pCam1300-CRISPR-B. Genes encoding Cas9p, gRNA-B, and hygromycin resistance (HygR) are represented by purple, green, and black arrows, respectively. The Cas9pmodule is shown in detail. Primers used to genotype Cas9p are marked on the map. c, Scheme for transformation, selection, and regeneration.« less
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