title: Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.
abstract: The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.|University of Chinese Academy of Sciences (CAS), Shanghai, China.
authors.forename: Zhengjing
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.|University of Chinese Academy of Sciences (CAS), Shanghai, China.|Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Zhengyan
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.|University of Chinese Academy of Sciences (CAS), Shanghai, China.
authors.forename: Pengliang
authors.initials: P
authors.lastname: Wei
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Agriculture and Food Sciences, University of Queensland, Brisbane, Qld, Australia.
authors.forename: José Ramón
authors.initials: JR
authors.lastname: Botella
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.|Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA.
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003433:Crosses, Genetic; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D060005:Genotyping Techniques; D005854:Germ Cells / Q000378:metabolism*; D055993:Germ Cells, Plant / Q000378:metabolism; D006824:Hybridization, Genetic; D040582:Inheritance Patterns / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics
keywords: Arabidopsis; CRISPR-Cas9; gene modification; germ-line-specific