ZFIN ID: ZDB-PUB-160725-28
Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology
De Santis, F., Di Donato, V., Del Bene, F.
Date: 2016
Source: Methods in cell biology   135: 171-88 (Chapter)
Registered Authors: Del Bene, Filippo, De Santis, Flavia
Keywords: CRISPR/Cas9, Clonal analysis, Cre recombinase, Genome editing, Tissue-specific knockout, Zebrafish
MeSH Terms:
  • Animals
  • Animals, Genetically Modified/genetics
  • CRISPR-Cas Systems/genetics*
  • Gene Deletion
  • Gene Knockout Techniques/methods*
  • Gene Transfer Techniques*
  • Genes, Reporter
  • Genetic Engineering/methods*
  • Genome
  • Green Fluorescent Proteins/genetics
  • Mutagenesis/genetics
  • Organ Specificity/genetics
  • Zebrafish/genetics
PubMed: 27443925 Full text @ Meth. Cell. Biol.
In the last few years the development of CRISPR/Cas 9-mediated genome editing techniques has allowed the efficient generation of loss-of-function alleles in several model organisms including zebrafish. However, these methods are mainly devoted to target-specific genomic loci leading to the creation of constitutive knock-out models. On the contrary, the analysis of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish. To circumvent this limitation, we present here a simple and versatile protocol to achieve tissue-specific gene disruption based on the Cas9 expression under the control of the Gal4/upstream activating sequence binary system. In our method, we couple Cas9 with green fluorescent protein or Cre reporter gene expression. This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells, and to follow them in vivo via reporter gene expression. Importantly, because none of the tools that we present here are restricted to zebrafish, similar approaches are readily applicable in virtually any organism where transgenesis and DNA injection are feasible.