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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Autores principales:	Yoshihiko Nanasato, Masafumi Mikami, Norihiro Futamura, Masaki Endo, Mitsuru Nishiguchi, Yasunori Ohmiya, Ken-ichi Konagaya, Toru Taniguchi
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/88d381bc5aa149be8e7f76311687a9fb
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spelling	oai:doaj.org-article:88d381bc5aa149be8e7f76311687a9fb2021-12-02T16:28:50ZCRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)10.1038/s41598-021-95547-w2045-2322https://doaj.org/article/88d381bc5aa149be8e7f76311687a9fb2021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-95547-whttps://doaj.org/toc/2045-2322Abstract 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.Yoshihiko NanasatoMasafumi MikamiNorihiro FutamuraMasaki EndoMitsuru NishiguchiYasunori OhmiyaKen-ichi KonagayaToru TaniguchiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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)
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.
format	article
author	Yoshihiko Nanasato Masafumi Mikami Norihiro Futamura Masaki Endo Mitsuru Nishiguchi Yasunori Ohmiya Ken-ichi Konagaya Toru Taniguchi
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 Portfolio
publishDate	2021
url	https://doaj.org/article/88d381bc5aa149be8e7f76311687a9fb
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CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

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