CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)
Abstract Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutag...
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doaj-88d381bc5aa149be8e7f76311687a9fb2021-08-15T11:23:23ZengNature Publishing GroupScientific Reports2045-23222021-08-0111111210.1038/s41598-021-95547-wCRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)Yoshihiko Nanasato0Masafumi Mikami1Norihiro Futamura2Masaki Endo3Mitsuru Nishiguchi4Yasunori Ohmiya5Ken-ichi Konagaya6Toru Taniguchi7Forest Bio-Research Center, Forestry and Forest Products Research InstituteGraduate School of Nanobioscience, Yokohama City UniversityDepartment of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research InstituteGraduate School of Nanobioscience, Yokohama City UniversityDepartment of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research InstituteDepartment of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research InstituteForest Bio-Research Center, Forestry and Forest Products Research InstituteForest Bio-Research Center, Forestry and Forest Products Research InstituteAbstract Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.https://doi.org/10.1038/s41598-021-95547-w |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yoshihiko Nanasato Masafumi Mikami Norihiro Futamura Masaki Endo Mitsuru Nishiguchi Yasunori Ohmiya Ken-ichi Konagaya Toru Taniguchi |
spellingShingle |
Yoshihiko Nanasato Masafumi Mikami Norihiro Futamura Masaki Endo Mitsuru Nishiguchi Yasunori Ohmiya Ken-ichi Konagaya Toru Taniguchi CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) Scientific Reports |
author_facet |
Yoshihiko Nanasato Masafumi Mikami Norihiro Futamura Masaki Endo Mitsuru Nishiguchi Yasunori Ohmiya Ken-ichi Konagaya Toru Taniguchi |
author_sort |
Yoshihiko Nanasato |
title |
CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) |
title_short |
CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) |
title_full |
CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) |
title_fullStr |
CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) |
title_full_unstemmed |
CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don) |
title_sort |
crispr/cas9-mediated targeted mutagenesis in japanese cedar (cryptomeria japonica d. don) |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2021-08-01 |
description |
Abstract Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome. |
url |
https://doi.org/10.1038/s41598-021-95547-w |
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