CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. 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 sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes 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 that CjACOS5 is 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.

CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.

A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.

Bone regeneration by modified gene-activated matrix: effectiveness in segmental tibial defects in rats.

Gene-activated matrix (GAM) is a matrix, such as collagen-containing plasmid vector, that encodes a protein to stimulate tissue regeneration. In the original GAM system, gene transfer efficiency was extremely low. We have recently reported that modifying GAM with calcium-phosphate precipitates (CaP) enhances the efficiency of gene transfer. The purpose of this study was to evaluate the effects of our modified GAM on tissue regeneration. We prepared critical size segmental bone defects in rat tibiae and transplanted GAM consisting of bovine atelocollagen and expression plasmid vector (bmp2), which encodes human BMP2, with or without CaP. The tibiae were later examined radiographically, histologically, and mechanically. Implantation of bmp2-CaP-collagen at 12 microg bmp2 bridged the bone defect at 4 weeks, and the strength of the bone was comparable to that of an intact tibia at 6 weeks. Implantation of bmp2-collagen at the same dose of bmp2 bridged the defect to a smaller extent. Neither collagen alone nor vacant vector-CaP-collagen bridged the defect. These results indicate that our modified GAM with CaP has the potential to be effective in tissue regeneration at lower plasmid DNA doses than used in previous studies.