title: High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease.
abstract: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination.
authors.affiliation: Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, King Abdullah University of Science and Technology, 4700, Thuwal, 23955-6900, Saudi Arabia.
authors.forename: Ayman
authors.initials: A
authors.lastname: Eid
authors.affiliation: Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, King Abdullah University of Science and Technology, 4700, Thuwal, 23955-6900, Saudi Arabia.
authors.forename: Zahir
authors.initials: Z
authors.lastname: Ali
authors.affiliation: Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, King Abdullah University of Science and Technology, 4700, Thuwal, 23955-6900, Saudi Arabia. magdy.mahfouz@kaust.edu.sa.
authors.forename: Magdy M
authors.initials: MM
authors.lastname: Mahfouz
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D005818:Genetic Engineering / Q000379:methods; D059765:Homologous Recombination; D006720:Homozygote; D008540:Meiosis / Q000235:genetics; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D029681:Arabidopsis Proteins; D004720:Endonucleases
keywords: Arabidopsis; CRISPR/Cas9; Genome editing; Genome engineering; Meiotic promoters; Targeted mutagenesis