title: Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.
abstract: Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.
authors.affiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045 Japan toshihisa.nomura@riken.jp sakaki@riken.jp.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Nomura
authors.affiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045 Japan.|Research and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi, 783-8502 Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045 Japan.|Faculty of Bioscience and Bioindustry, Tokushima University, 2-1 Josanjima, Tokushima, 770-8513 Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Osakabe
authors.affiliation: Faculty of Bioscience and Bioindustry, Tokushima University, 2-1 Josanjima, Tokushima, 770-8513 Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Osakabe
authors.affiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045 Japan toshihisa.nomura@riken.jp sakaki@riken.jp.|Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakakibara
mesh_terms: D044002:Bryophyta / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011523:Protoplasts; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D018749:RNA, Plant; D004720:Endonucleases
keywords: Bryophyte; CRISPR/Cas9 system; Genome editing; Physcomitrella patens; RNA-guided nucleases; Scopelophila cataractae