ZeGenesis – Genetic Services

BY
OUR
PLATFORMS

knock-out-lines-icon Knock-out lines

Gene knock-out (KO) is a genetic technique employed to inactivate one of an organism’s genes. The creation of stable KO zebrafish lines ensures to obtain isogenic lines in which every fish carries the same mutation in the gene of interest. This approach is the best option if the candidate target gene has already been identified and the aim of the experiment is to achieve a fine characterization of its biological function, generate a disease model or perform a drug screening.

This technique can be used for generating single KO lines (mutation of one gene) or double KO lines (mutation of two genes, e.g. two paralogues).

According to your needs, either F1 heterozygous KOs (carrying one mutated allele) or F2 homozygous KOs (with two mutated alleles) can be generated and shipped worldwide.

Applications

  • Study the role of a gene by evaluating loss-of-function phenotypes
  • Modelling of human genetic diseases for drug-screening

Advantages

Highly efficient mutagenesis (close to 100%)

Extreme flexibility of the CRISPR/Cas9 system that allows customized generation of zebrafish lines

High rate of success in KO generation (99%)

Method description

We are an officially licensed CRISPR solutions provider. We design sgRNAs having high specificity and efficiency to guarantee a mutagenesis rate close to 100%.

A specific sgRNA guides the Cas9 endonuclease to the desired locus of interest to induce targeted double-strand breaks. Error-prone mechanisms of DNA-repair can cause the generation of small insertions and deletions (INDELs) potentially disrupting the coding sequence of the targeted gene and promoting the formation of loss-of-function mutations.

Figure 1. Editing a gene using the CRISPR/cas9 technique. The system is based on 2 components: agene-specific sgRNA and a  Cas9 endonuclease. sgRNA and Cas9 form a complex which binds on a specific sequence of the genome and generates a double-strand break followed by DNA repair.
Figure 2. Pipeline for the generation of stable F1 and F2 KO using the CRISPR/Cas9 technique.
  1. To generate mutant alleles in zebrafish, a complex formed by the Cas9 and the sgRNA designed against the selected gene is injected into wild-type one-cell-stage embryos (F0).
  2. Once they reach sexual maturity, injected larvae (F0) are screened to identify a founder carrying a loss-of-function mutation in the germline.
  3. The selected founder is then crossed to a wild-type fish, thus generating a population of heterozygous and wild-type offspring (F1).
  4. The inbreeding of adult heterozygous fish (F1) gives rise to homozygous mutant, heterozygous and wild type larvae (F2), in which the phenotypes deriving from gene inactivation can

Deliverables

  • Heterozygous transgenic embryos (F1)
  • Homozygous transgenic embryos (F2)

References

  1. Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 2014 Jan;24(1):125-31.