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 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.

Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca

“…Another possible cause that resulted in the chimeric mutation might be the culture temperature applied in this research, which was significantly lower than the most suitable temperature, about 37°C, for CRISPR/Cas9 system (Xiang et al, 2017 ). A high frequency of chimerism during genome editing in conifers was also reported by Poovaiah et al ( 2021 ) and Nanasato et al ( 2021 ). Poovaiah et al ( 2021 ) thought the delayed Cas9 cleavage in the proliferating embryogenic tissue was the reason for chimerism, while Nanasato et al ( 2021 ) thought the ongoing function of CRISPR/Cas9 led to chimerism.…”

“…A high frequency of chimerism during genome editing in conifers was also reported by Poovaiah et al ( 2021 ) and Nanasato et al ( 2021 ). Poovaiah et al ( 2021 ) thought the delayed Cas9 cleavage in the proliferating embryogenic tissue was the reason for chimerism, while Nanasato et al ( 2021 ) thought the ongoing function of CRISPR/Cas9 led to chimerism. The exact reason for chimerism in this research was unclear, although the active division of embryogenic cells and the unfavorable temperature for Cas9 activity might be the cause.…”

“…With well-established systems of stable transformation and plant regeneration, techniques based on CRISPR/Cas9 could be used commonly in coniferous species in the near future. Interestingly, there were no large deletions between the two target sites in this research, whereas large deletions between two target sites were reported both in Pinus radiata (Poovaiah et al, 2021 ) and Cryptomeria japonica (Nanasato et al, 2021 ). Since only one gene was edited in this research, we could not make a conclusion on large deletions at other sites of the genome.…”

“…It would also be efficient in Larix according to the function of the PaU6 promoter in this genus. Additionally, the recently published reports on genome editing in conifers (Nanasato et al, 2021 ; Poovaiah et al, 2021 ) have indicated that some improvements on the CRISPR/Cas9 toolbox are worthy to be carried out in further studies, such as using the Ubi promoter to drive the expression of Cas9 and/or testing some other U6 promoters.…”

“…However, no genome-edited plants were regenerated in this research (Ren Q. et al, 2021 ). Most recently, CRISPR/Cas9-mediated-targeted mutagenesis has been reported in both Pinus radiata (Poovaiah et al, 2021 ) and Cryptomeria japonica (Nanasato et al, 2021 ), which indicated the feasibility of applying CRISPR/Cas9 in conifers. These reports were very exciting, but a high frequency of chimera was found in both of the studies.…”