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knock-out-lines-icon CRISPR Knockout Service in Zebrafish

CRISPR knockout zebrafish

ZeClinics is at the forefront of genetic engineering, leveraging the groundbreaking CRISPR Cas9 gene editing technique. We excel in delivering highly accurate and efficient zebrafish CRISPR knockout solutions for precise gene editing.

Utilizing cutting-edge CRISPR Cas9 technology, we ensure reliable genetic modifications in zebrafish. Our expert scientists offer comprehensive support throughout the entire process, from sgRNA design, to the generation and validation of zebrafish knockout mutants.

Why Choose ZeClinics's Zebrafish CRISPR Cas9 Knockout Services?

Gene knockouts are a commonly used tool for biologists to understand gene function. Observing phenotypic changes following gene deletion can reveal valuable insights regarding the gene's role within the organism.

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

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

CRISPR Cas9 Gene Knockout

Get the gene editing you want along with the support you need to start your gene knockout project.

Comparison of Knockouts vs Crispants

If speed is what you need, ZeClinics' CRISPANTS offer a fast-track option for screening loss-of-function alleles in vivo.

crispr knockouts versus crispants

Applications of Knockout Zebrafish Lines

  • Disease modeling: our customized zebrafish knockout solutions help you to faithfully establish loss-of-function human disease models.
  • Target validation: the generation of zebrafish knockout mutants is the best option for a fine characterization of a gene’s biological function.
  • Drug screening: in vivo efficacy testing of candidate therapeutic compounds in the context of a disease, to accelerate your compound development.

Key Benefits of CRISPR Gene Knockout in Zebrafish

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 knockout generation (99%)

CRISPR Gene Knockout 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.

CRISPR/cas9 technique
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.
CRISPR knockout generation process in zebrafish
Figure 2. Pipeline for the generation of stable F1 and F2 KO using the CRISPR/Cas9 technique.

Zebrafish Knockout Service Options

CRISPR knockout service options

Service Deliverables

F0 Mosaic Knockouts

Choose this option if you wish to outsource the design and validation of a CRISPR-based knockout. You will obtain F0 mutant larvae ready to grow in your facility. This is a good option for researchers who want to entrust skilled genome editing experts with the most delicate phases of a zebrafish knockout.

Experimental steps
  • Genome editing strategy designed by an experienced genetic engineer 
  • In silico design of the sgRNAs
  • In vivo evaluation of the sgRNAs’ cutting efficiency
  • Injection of validated Cas9/sgRNAs complexes into one-cell-stage embryos
Optional assessment
  • Sequencing of your target locus to detect potential polymorphisms prior to the sgnRNAs’ design
Deliverables
  • Batch of injected larvae to grow in your own facility
  • Validated genotyping protocol

F1 Isogenic Knockouts

Select this option if you desire us to deliver the offspring of a validated founder (F1 larvae). This is an excellent choice for researchers who, in addition to leveraging our expertise in designing an efficient knockout approach, seek to delegate the labor-intensive phase of founder screening and identification.

Experimental steps
  • Steps listed in “F0 MOSAIC KNOCKOUTS” plus:
  • Growth of injected F0 larvae until adulthood 
  • Founder screening
  • Sequencing and validation of the knockout allele
  • Breeding of the selected founder
  • Maintenance of the selected founder for 4 months
Deliverables
  • Batch of F1 larvae to grow and genotype in your own facility
  • Validated genotyping protocol

F2 Isogenic Knockouts

Select this option if you want us to provide a set of siblings (wild-type, heterozygous, and homozygous) from an isogenic knockout line. This is an ideal choice for researchers seeking to rely on our extensive expertise to perform the entire process of developing a new line.

Experimental steps
  • Steps listed in “F1 ISOGENIC KNOCKOUTS" plus:
  • Growth of F1 larvae until adulthood
  • Fish genotyping
  • Crossing to generate F2 offspring
  • Maintenance of the F1 for 4 months
Deliverables
  • Batch of F2 larvae to grow and genotype in your own facility
  • Validated genotyping protocol

F3 Isogenic Knockouts

Opt for this solution if you desire to receive a homogeneous population of homozygous mutants. This is the optimal choice if the induced mutation is non-lethal and you are interested in analyzing homozygous fish.

Experimental steps
  • Steps listed in “F2 ISOGENIC KNOCKOUTS” PLUS:
  • Growth of F2 larvae until adulthood
  • Fish genotyping
  • Crossing to generate F3 homozygous offspring
  • Maintenance of the F2 for 4 months
Deliverables
  • Batch of F3 larvae to grow in your own facility
  • Validated genotyping protocol

We'd like to hear from you

If you want more information about our CRISPR knockout
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.