Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9

Research output: Contribution to journalResearch articleContributedpeer-review

Abstract

New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER T2 upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER T2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER T2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

Details

Original languageEnglish
Pages (from-to)41-50
Number of pages10
JournalCell and tissue research
Volume372
Issue number1
Publication statusPublished - Apr 2018
Peer-reviewedYes

External IDs

Scopus 85041927406
ORCID /0000-0003-0283-0211/work/142257332

Keywords

ASJC Scopus subject areas

Keywords

  • Animals, Base Sequence, CRISPR-Associated Protein 9/metabolism, CRISPR-Cas Systems/genetics, Gene Expression, Gene Knock-In Techniques, Gene Targeting, Genetic Loci, Integrases/metabolism, Mesencephalon/cytology, Otx Transcription Factors/genetics, Prosencephalon/cytology, Recombination, Genetic/genetics, Zebrafish/genetics, Zebrafish Proteins/genetics

Library keywords