ZeGenesis – Genetic Services

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crispants-icon F0 Knock-outs (Crispants)

The injection of zebrafish one-cell-stage embryos with Cas9/sgRNAs complexes makes it possible to induce genomic mutations at the desired site with very high efficiency (up to 100%).

Thanks to this extremely high performance, at ZeClinics we are able to generate somatic mutants (Crispants) carrying a high rate of biallelic mutations in F0 animals. This approach ensures the creation of “de facto” F0 mutants enabling an exhaustive evaluation of loss-of-function phenotypes in a short time (2-3 months). Genotyping of mosaic F0 larvae permits a fine genotype/phenotype correlation, enabling the achievement of extremely reliable results. In addition, a Crispants-based approach is the ideal system to evaluate, in vivo, the activity of new tools (like modified or improved Cas9 proteins).

Applications

  • Cost-effective screening of candidate genes that are suspected to be involved in a given disease
  • Rapid evaluation of loss-of-function phenotypes 
  • Evaluation, in vivo, of the safety, efficacy, and efficiency of new genome editing tools

Advantages

High throughput screening of multiple candidate genes

Fast target validation prior to the generation of stable KO

Non-invasive, in vivo readouts to detect KO-induced phenotypes at the level of cells, tissues or organs 

Unlike larvae injected with morpholino or antisense oligonucleotides, Crispants do not recover a normal gene expression after the first days of development

Precise genotype/phenotype correlation 

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.

To generate mutant alleles in zebrafish, the Cas9 and the sgRNA(s) designed against the selected gene are injected into wild-type one-cell-stage embryos (F0). Mutagenesis takes place during early stages of embryonic development, giving rise to F0 larvae carrying a mosaic set of somatic and germline mutations. High mutagenesis rates in somatic KOs enable the evaluation of the resulting loss-of-function phenotypes already in F0 larvae.

Phenotypes resulting from the genetic modification can be characterized on demand in the whole organism or in specific organs.

Figure 1. Generation and analysis of somatic F0 KO (Crispants) using the CRISPR/cas9 technique. Upon injection with highly efficient sgRNA/Cas9 complexes, expected phenotypes can be readily analyzed in F0 KO larvae. 

Readouts

Results of the phenotypic analysis according to customers' interests and needs:

  • Teratogenic phenotypes
  • Organ-specific morphological or functional defects (heart-specific, liver-specific etc.)
  • Behavioral phenotypes

References

  1. Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development. 2016 Jun 1;143(11):2025-37.