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.
Highlights
To obtain the desired trait through gene modification in a short time, programmable nuclease-mediated targeted mutagenesis is the option of choice and several methods, such as zinc-finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 system (CRISPR/Cas9), have been developed
To determine the efficiency of the CRISPR/Cas[9] system in C. japonica, we first obtained an optimal promoter for driving the expression of SpCas[9]
We selected target sites that have been previously reported[37,38,39] (Table 1). pZK_FFCas[9] vectors containing the gRNA of green fluorescent protein (GFP) target #1 driven by 13 different U6 promoters were introduced into N4, and 17–138 transgenic embryogenic tissue (ET) lines were obtained for each vector (Table 2) by screening in a medium with 25 mg/L kanamycin
Summary
Development of “male-sterile” C. japonica trees is a breeding target in Japan. The use of CRISPR/Cas[9] system, first reported in 2 01212, has exploded because of its efficiency and simplicity, and has been applied to at least 45 plant genera[13]. CRISPR/Cas9-mediated targeted mutagenesis in Populus, which is a model woody species, was first reported in 2 01521,22. We demonstrated CRISPR/Cas9-mediated targeted mutagenesis in C. japonica through Agrobacterium-mediated transformation. As a case study, we attempted to disrupt an endogenous gene, namely magnesium chelatase subunit I (CjChlI) that is required for chlorophyll b iosynthesis[23,24] Disruption of this gene results in an albino or pale green phenotypes, which readily confirms CRISPR/Cas9-mediated targeted mutagenesis.
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