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PLoS One. 2016 Mar 1;11(3):e0150188. doi: 10.1371/journal.pone.0150188. eCollection 2016.

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

Author information

1
Department of Neurosciences, Research Centre of the University of Montréal Hospital Centre, Montréal, Québec, Canada.
2
Department of Neurology and Neurosurgery, Research Institute of the McGill University Health Centre and Centre for Research in Neuroscience, Montréal, Québec, Canada.

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.

PMID:
26930076
PMCID:
PMC4773037
DOI:
10.1371/journal.pone.0150188
[Indexed for MEDLINE]
Free PMC Article

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