title: RNA-guided Cas9 as an in vivo desired-target mutator in maize.
abstract: The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.
authors.forename: Chuxi
authors.initials: C
authors.lastname: Li
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.
authors.forename: Changlin
authors.initials: C
authors.lastname: Liu
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.|Anhui Agricultural University, Hefei, Anhui Province, China.
authors.forename: Xiantao
authors.initials: X
authors.lastname: Qi
authors.affiliation: AgGene Bio-tech Seed Industry Group, Shenzhen, China.
authors.forename: Yongchun
authors.initials: Y
authors.lastname: Wu
authors.affiliation: AgGene Bio-tech Seed Industry Group, Shenzhen, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Fei
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.
authors.forename: Long
authors.initials: L
authors.lastname: Mao
authors.affiliation: Anhui Agricultural University, Hefei, Anhui Province, China.
authors.forename: Beijiu
authors.initials: B
authors.lastname: Cheng
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.
authors.forename: Xinhai
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, China.
authors.forename: Chuanxiao
authors.initials: C
authors.lastname: Xie
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing; D040582:Inheritance Patterns; D009154:Mutation; D059645:Mutation Rate; D000069600:Plant Breeding / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics; D017422:Sequence Analysis, DNA; D003313:Zea mays / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D004720:Endonucleases; D000094704:RNA, Guide, CRISPR-Cas Systems; C000606107:Cas9 endonuclease Streptococcus pyogenes
keywords: desired-target mutator; genome editing; sexual plant breeding