CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish.
The targeted introduction of mutations utilizing sequence specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (RNA-guided nucleases, RGNs) has revolutionized reverse genetic approaches in numerous model organisms. In zebrafish, both systems were successfully applied to generate loss-of-function alleles by targeting open reading frames or deletion and inversion of whole chromosomal regions. In addition to the production of these loss-of-function alleles, genomic engineering by insertion of short sequences utilizing single stranded DNA oligonucleotides as templates for homology based repair was made possible, enabling effective insertion of loxP sites or tags for protein coding genes. Recent studies based on homologous recombination and non-homologous end joining have also broadened the repertoire for genome editing. These approaches allow the targeted insertion of open reading frames or even whole donor vectors. In this review we summarize the use of TALENs and RNA-guided nucleases in the field of zebrafish genetics with a special focus on knock-in approaches.
Institut Curie, Centre de Recherche, Paris F-75248, France; CNRS UMR 3215, Paris F-75248, France; INSERM U934, Paris F-75248, France; Centre for Organismal Studies Heidelberg, Im Neuenheimer Feld 230, University of Heidelberg, 69120 Heidelberg, Germany. Electronic address: thomas.auer@curie.fr.
Thomas O
TO
Auer
Institut Curie, Centre de Recherche, Paris F-75248, France; CNRS UMR 3215, Paris F-75248, France; INSERM U934, Paris F-75248, France. Electronic address: filippo.del-bene@curie.fr.
Filippo
F
Del Bene
D000595:Amino Acid Sequence; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
D029961:Zebrafish Proteins
CRISPR/Cas9; Homologous recombination; Homology independent repair; TALENs; Zebrafish