CRISPR/Cas9-mediated disruption ofCjACOS5confers no-pollen formation on sugi trees (Cryptomeria japonicaD. Don)

Abstract

Sugi (Cryptomeria japonicaD. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugiCjACOS5gene is an ortholog ofArabidopsis ACOS5and riceOsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutatedCjACOS5using the CRISPR/Cas9 system. As a result of sugi transformation mediated byAgrobacterium tumefaciensharboring theCjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-typeCjACOS5genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest thatCjACOS5is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.