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 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 3.1–41.4% 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 102 of 103 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out endogenous C. japonica magnesium chelatase subunit I (CjChlI) using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in genome-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.

A chromosome-level genome assembly of a model conifer plant, the Japanese cedar,Cryptomeria japonicaD. Don

“…japonica can be used as a model conifer for several reasons. The species is easily propagated by cutting [ [61,62], flowered by plant hormones like gibberellic acid [63], transformed by agrobacterium [64], and can be edited by CRISPR/Cas9 [65]. This study aims at providing the highquality, chromosome-level genome sequence of the C. japonica.…”