title: Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.
abstract: CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Jinjie
authors: J
authors: Zhu
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Ning
authors: N
authors: Song
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Silong
authors: S
authors: Sun
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Weilong
authors: W
authors: Yang
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Haiming
authors: H
authors: Zhao
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
authors: Weibin
authors: W
authors: Song
authors: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Electronic address: jlai@cau.edu.cn.
authors: Jinsheng
authors: J
authors: Lai
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D004706:Endodeoxyribonucleases / Q000378:metabolism; D000072669:Gene Editing; D018745:Genome, Plant; D040582:Inheritance Patterns; D016296:Mutagenesis; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics; D014158:Transcription, Genetic; D003313:Zea mays / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D004706:Endodeoxyribonucleases
keywords: CRISPR-Cas9; Heritability; Maize; Targeted mutagenesis