Rapid Zebrafish Genetic Model Creation Using Crispants

What is Crispant?

Crispant is a new CRISPR/Cas9 method generates F0 mutant zebrafish that cuts costs, time, and reduces animal use for researchers interested in screening loss-of-function alleles in vivo. Using the F0 knockout method in zebrafish, it takes as little as a week to go from gene disruption to behavioral phenotype instead of more than six months using standard genetic approaches. The Crispant approach is an efficient tool for disease modeling, proof-of-concept studies, and lead target validation. 

Using traditional CRISPR approaches to create a stable knockout model typically takes 9-12 months, making it incompatible with the fast pace of discovery of new genetic associations.

To combat the long lead times associated with traditional genetic models, InVivo Biosystems optimized the Crispant approach to create F0 knock-out animals by “breaking” a gene in multiple locations.

The transient knock-out line is generated in 1 month or less and can be used to directly create stable zebrafish knock-out mode

  • Save time on go no-go decisions: Decide in less than a month whether a functional genetic model can be created in vivo. Assess multiple genetic targets in vivo in a fraction of the time.
  • Get a head start on full model creation while collecting early data: Transient zebrafish created with this technique can be used to create the stable zebrafish model.
  • Quick evaluation of the biological function of a single gene or a set of genes in concert
  • Gene lethality assessment
  • Screening to determine if a stable mutation should be created
  • Rapidly assay candidate targets for your therapeutic
  • Targeting specific functional regions of your gene of interest
  • Crispant Verified Injection Mix: inject-ready mix containing 3 sgs and Cas9 enzyme.
    Identify 5 sgs across the gene of interest, test in vivo cutting efficiency; select the verified 3 best cutters.

  • Crispant In Silico Predicted Injection Mix: inject-ready mix containing 3 sgs and Cas9 enzyme.
    Identify 3 sgs across the gene of interest based on predicted cutting.

Replicating Heartstrings(tbx5a) mutant using crispant technique. Knock-out created in less than 1 month.

Fig. 1: See Crispant in action.

Fig. 2: Heartstrings Mutant (tbx5). Crispant phenocopies both the stable KO and the morphant showing (1) slowing of heartbeat, (2) long stretched out heart, and (3) partial or full loss of pectoral fins.

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