ZeGenesis – Genetic Services

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knock-out-lines-icon Knock-out lines

As an officially CRISPR licensed provider, ZeClinics can permanently inactivate your gene of interest in zebrafish to generate tailored knock-out (KO) lines from 6/7 months. This approach is the best option for a fine characterization of a gene’s biological function, for disease modeling, and for drug screening in the context of a disease.

Using the CRISPR/Cas9 system in zebrafish, we can disrupt the CDS of a gene or the integral regulatory domains (KO null), or remove the entire functional domain or the entire CDS depending on gene size (KO deletion).

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

CRISPR-Cas9 Gene Knock-out

Get the edit you want along with the support you need to start your KO project.

However, if speed is what you're after, ZeClinics' Crispants offer a fast-track option for target validation.

Applications

  • Target validation: fine characterization of a gene’s biological function
  • Disease modeling
  • Drug screening studies in the context of a disease

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 design sgRNAs having high specificity and efficiency to guarantee a mutagenesis rate close to 100%.

Specific sgRNA guide 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. Alternatively, a sequence of interest can also be completely deleted.

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.
  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 line can be propagated up to F2 or F3 generations on demand.
  5. The phenotypes deriving from gene inactivation can be analyzed in F1, F2, and F3 generations.
Figure 2. Pipeline for the generation of stable F1 and F2 KO using the CRISPR/Cas9 technique.

Deliverables

  • The sequence of sgRNAs and primers
  • Protocol for genotyping PCR
  • Genomic information of the founder carrying the selected mutation
  • Changes that the INDELs cause at protein level (KO null)
  • F1, F2, or F3 embryos:
Generation of F3 embryos is only possible if the mutant is adult viable.
WT, wild-type; HET, heterozygous; HOM, homozygous.

We'd like to hear from you

If you want more information about our knock-out
generation services or have any other questions,
please contact our experts.

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.