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ZeGenesis – Genetic Services

knock-in-lines-icon Knock-in lines

As an officially CRISPR licensed provider, ZeClinics can generate tailored knock-in (KI) lines by introducing an exogenous sequence into a specific locus of the zebrafish genome (site-directed mutagenesis).

Using the CRISPR/Cas9 system in zebrafish, we can insert short nucleotide sequences (like point mutations, loxP sites, or protein tags) or long sequences of exogenous DNA (like reporter genes or mutated genes’ CDSs).

This strategy is often used to understand gene function, to more precisely model human diseases, or to establish genetic tools to support phenotypic analysis.

CRISPR-Cas9 Gene Knock-in

Get the edit you want along with the support you need to start your KI project.

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Compared to Tol2 transgenesis, CRISPR/Cas9 is less efficient but much more precise. Unlike, overexpressing genes by mRNA injection, KI induces stable and permanent edits in the zebrafish genome.

Applications

  • Introduce point mutations (humanized zebrafish)
  • Introduce fluorescent tag at the native locus
  • Insert protein tags
  • Insert LoxP sites
  • Generate fusion proteins

Advantages

Targeted integration of the exogenous sequence of interest into the zebrafish genome

Extremely accurate recapitulation of the expression patterns of the target genes

Produce disruption in WT sequence

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

Initial phase:

  • The sequence of sgRNA employed
  • The sequence of the donor plasmid

Confirmation phase:

  • Pictures of reporter protein expression in comparison with the expected expression pattern
  • Sequencing results of a KI-specific PCR at the integration site
  • Percentage of larvae showing expression of the fluorescent reporter (% KI efficiency)

Generation phase:

  • The sequence of the KI allele identified in the founder
  • F1, F2, or F3 embryos:
Generation of F3 embryos is only possible if the mutant is adult viable. WT, wild-type; HET, heterozygous; HOM, homozygous.

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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.