Improvement of genome editing efficiency by Cas9 codon optimization in Japanese cedar (Cryptomeria japonica D. Don)

Japanese cedar or sugi (Cryptomeria japonica D. Don) is among the most important plantation conifers in Japan, occupying 12% of the total land area in the country. We have successfully established a CRISPR/Cas9-based genome editing system in C. japonica. However, in practical use, we encountered problems of low efficiency when generating biallelic mutations, i.e., target gene knockouts. As part of our efforts to improve efficiency, we codon-optimized the Cas9 gene, evaluated by the genome editing efficiency of CjChl I, a gene encoding a chlorophyll biosynthesis enzyme. As a result, our codon-optimized SpCas9, named ^CjSpCas9, performed the highest genome editing efficiency of two targets (t4, t1+t2). Specifically, the biallelic disruption efficiency of the CjChl I with ^CjSpCas9 was 1.8-fold higher than that of the SpCas9 gene optimized for Arabidopsis thaliana (^AtSpCas9) and 2.0-fold higher than that of the SpCas9 gene optimized for Orysa sativa (^OsSpCas9) for t4, respectively. For t1+t2, the efficiency was 4.9-fold higher than that of ^AtSpCas9 and 1.4-fold higher than that of ^OsSpCas9, respectively. Our western blotting analysis proved that the Cas9 protein accumulation increased upon codon frequency optimization. We concluded that the observed efficiency improvement was due to the increased Cas9 protein quantity. The efficient genome editing system we report here would accelerate molecular breeding in conifers.