ZFIN ID: ZDB-PUB-160302-4
Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System
Armstrong, G.A., Liao, M., You, Z., Lissouba, A., Chen, B.E., Drapeau, P.
Date: 2016
Source: PLoS One   11: e0150188 (Journal)
Registered Authors: Drapeau, Pierre
Keywords: Point mutation, Zebrafish, Sequence motif analysis, Non-homologous end joining, Polymerase chain reaction, Embryos, Missense mutation, Restriction fragment length polymorphism analysis
MeSH Terms:
  • Amino Acid Sequence
  • Amyotrophic Lateral Sclerosis/genetics*
  • Animals
  • Base Sequence
  • CRISPR-Cas Systems/genetics*
  • DNA Repair
  • DNA, Single-Stranded
  • DNA-Binding Proteins/genetics*
  • Disease Models, Animal
  • Gene Knock-In Techniques/methods
  • Humans
  • Oligodeoxyribonucleotides/genetics
  • Point Mutation*
  • Polymorphism, Single Nucleotide
  • RNA-Binding Protein FUS/genetics*
  • Reproducibility of Results
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Zebrafish/genetics
  • Zebrafish Proteins/genetics*
PubMed: 26930076 Full text @ PLoS One
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.