ZeGenesis – Genetic Services

Crispants: Fast-track Knock-outs

ZeClinics' Crispants are a fast-track choice for target validation and disease modeling. These somatic F0 mutants, which carry a high rate of biallelic mutations, enable a high throughput evaluation of loss-of-function phenotypes in a really short time (2-3 months).

Unlike other alternatives for fast target validation, such as morpholino or ASO knock-downs, the stable genetic modifications of Crispants allow studying biological processes at later stages.

Crispants are also interesting to confirm the expected phenotypic output of your custom mutation before establishing a KO line for further studies, such as drug screening.

Applications

• Fast access to gene function (functional genomics): rapid evaluation of loss-of-function phenotypes.
• Fast target validation: screening of several candidate genes.
• Fast disease modeling.

Advantages

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

Scalable solution: high throughput screening of multiple candidate genes

Confirmation of the phenotypic output before establishing the KO line

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

Method description

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 the early stages of embryonic development, giving rise to F0 larvae carrying a mosaic set of somatic and germline mutations.

Crispants carrying high rates of mutations in the selected gene are phenotypically preselected. These high mutagenesis rates in somatic KOs enable the evaluation of the resulting loss-of-function phenotypes already in F0 larvae.

Readouts

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

• Teratogenic phenotypes
• 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.