title: Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System.
abstract: The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.
authors.affiliation: Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
authors.forename: Gary Alan Barclay
authors.initials: GA
authors.lastname: Armstrong
authors.affiliation: Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
authors.forename: Meijiang
authors.initials: M
authors.lastname: Liao
authors.affiliation: Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: You
authors.affiliation: Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lissouba
authors.affiliation: Department of Neurology and Neurosurgery, Research Institute of the McGill University Health Centre and Centre for Research in Neuroscience, Montréal, Québec, Canada.
authors.forename: Brian Edwin
authors.initials: BE
authors.lastname: Chen
authors.affiliation: Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Drapeau
mesh_terms: D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004260:DNA Repair; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics*; D004195:Disease Models, Animal; D055879:Gene Knock-In Techniques / Q000379:methods; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D034702:RNA-Binding Protein FUS / Q000235:genetics*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D034702:RNA-Binding Protein FUS; C585345:Tardbp protein, zebrafish; D029961:Zebrafish Proteins