Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

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...

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Bibliographic Details
Published in:PloS one 2016-03, Vol.11 (3), p.e0150188-e0150188
Main Authors: Armstrong, Gary Alan Barclay, Liao, Meijiang, You, Zhipeng, Lissouba, Alexandra, Chen, Brian Edwin, Drapeau, Pierre
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amyotrophic lateral sclerosis
Amyotrophic Lateral Sclerosis - genetics
Analysis
Animal models
Animals
Base Sequence
Biology and Life Sciences
CRISPR
CRISPR-Cas Systems - genetics
Danio rerio
Development and progression
Disease Models, Animal
DNA Repair
DNA, Single-Stranded
DNA-Binding Proteins - genetics
Drosophila
Editing
FUS protein
Gene Knock-In Techniques - methods
Gene mutation
Genes
Genetic aspects
Genomes
Homology
Humans
Mutation
Neurosciences
Non-homologous end joining
Nucleotides
Oligodeoxyribonucleotides - genetics
Physiological aspects
Point Mutation
Polymorphism, Single Nucleotide
Reproducibility of Results
Research and Analysis Methods
RNA-Binding Protein FUS - genetics
Rodents
Sequence Homology, Amino Acid
Sequence Homology, Nucleic Acid
Single-nucleotide polymorphism
Zebrafish
Zebrafish - genetics
Zebrafish Proteins - genetics
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0150188