ZeGenesis – Genetic Services

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knock-in-lines-icon Knock-in lines

Gene knock-in (KI) is the process of insertion of an exogenous sequence into a specific locus of the genome. We use it to introduce stable genetic modifications in the zebrafish genome in order to generate complex disease models and tools boosting their phenotypic characterization.

This technique can be used for introducing in the zebrafish genome short nucleotide sequences (like point mutations, loxP sites or protein tags)  or long sequences of exogenous DNA (like reporter genes or mutated genes’ CDSs).

According to your needs, either F1 heterozygous KIs (carrying one exogenous allele) or F2 homozygous KIs (with two exogenous alleles) can be generated and shipped worldwide.

Applications

  • Validate the pathogenic role of candidate genes
  • Create disease models for drug-screening
  • Establish genetic tools to support phenotypes’ analysis 
  • Generation of customized reporter lines

Advantages

Highly efficient and precise integration of the exogenous sequence of interest into the zebrafish genome.

Extremely accurate recapitulation of expression patterns of sequences of interest compared to random transgenesis.

Method description

To obtain the targeted insertion of an exogenous DNA sequence, one cell stage zebrafish embryos are injected with Cas9/sgRNA complexes targeting the gene of interest and a donor DNA containing the sequence of the KI allele. Successful integration can be proved either by deep sequencing, KI-specific PCR or by expression of fluorescent reporters. Injected fish are grown to sexual maturity and subsequently screened to identify a founder carrying the KI allele in its germline.

Figure 1. Generation of larvae carrying a KI allele using the CRISPR/Cas9 technique. Upon injection of the Cas9/sgRNA complex, in the confirmation phase, larvae are screened for integration of the sequence of interest by sequencing or expression of a fluorescent reporter.
Figure 2. Proof of principle of GFP integration into zebrafish endogenous loci in F0 animals (confirmation phase). Left Panel: integration of GFP in locus with specific expression in a developing heart. Central Panel: Integration of GFP in a locus with a specific expression in the hindbrain. Right Panel: Widespread expression of GFP in motoneurons.

Deliverables

  • Mosaic F0 embryos (Confirmation Phase)
  • Heterozygous KI embryos (F1
  • Homozygous KI embryos (F2)

References

  1. Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 2014 Jan;24(1):142-53.
  2. Li J, Zhang B, Bu J, Du J. Intron-based genomic editing: a highly efficient method for generating knockin zebrafish. Oncotarget. 2015 Jul 20;6(20):17891-4.
  3. Kimura, Y., Hisano, Y., Kawahara, A. et al. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Sci Rep 4, 6545 (2014).
  4. Albadri S, De Santis F, Di Donato V, Del Bene F. CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish. 2017 Sep 15. In: Jaenisch R, Zhang F, Gage F, editors. Genome Editing in Neurosciences [Internet]. Cham (CH): Springer; 2017.
  5. Albadri S, Del Bene F, Revenu C. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods. 2017 May 15;121-122:77-85.