title: Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System.
abstract: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.
authors: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Chao
authors: C
authors: Feng
authors: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Jing
authors: J
authors: Yuan
authors: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Rui
authors: R
authors: Wang
authors: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Yang
authors: Y
authors: Liu
authors: Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA.
authors: James A
authors: JA
authors: Birchler
authors: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: fphan@genetics.ac.cn.
authors: Fangpu
authors: F
authors: Han
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D004706:Endodeoxyribonucleases / Q000378:metabolism; D000072669:Gene Editing; D018390:Gene Targeting; D017343:Genes, Plant; D005819:Genetic Markers; D018745:Genome, Plant; D006570:Heterochromatin / Q000378:metabolism; D003313:Zea mays / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D005819:Genetic Markers; D006570:Heterochromatin; D004706:Endodeoxyribonucleases
keywords: CRISPR/Cas9; Heterochromatic region; Maize; Targeted genome modification