title: Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish.
abstract: With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc. but also ways to circumvent encountered issues with HDR insertions by the development of non-homologous dependent strategies. While imprecise, these homology-independent mechanisms based on non-homologous-end-joining (NHEJ) repair have been employed in zebrafish to generate reporter lines or to accurately edit an open reading frame by the use of intron-targeting modifications. Therefore, with higher efficiency and insertion rate, NHEJ-based knock-in seems to be a promising approach to target endogenous loci and to circumvent the limitations of HDR whenever it is possible and appropriate. In this perspective, we propose new strategies to generate cDNA edited or tagged insertions, which once established will constitute a new and versatile toolbox for CRISPR/Cas9-based knock-ins in zebrafish.
authors.affiliation: Institut Curie, PSL Research University, INSERM U934, CNRS UMR3215, 75248 Paris Cedex 05, France.
authors.forename: Shahad
authors.initials: S
authors.lastname: Albadri
authors.affiliation: Institut Curie, PSL Research University, INSERM U934, CNRS UMR3215, 75248 Paris Cedex 05, France. Electronic address: filippo.del-bene@curie.fr.
authors.forename: Filippo
authors.initials: F
authors.lastname: Del Bene
authors.affiliation: Institut Curie, PSL Research University, INSERM U934, CNRS UMR3215, 75248 Paris Cedex 05, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Revenu
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004625:Embryo, Nonmammalian; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D016678:Genome; D008845:Microinjections; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair; D015027:Zebrafish / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9; Genome editing; Homologous recombination; Knock-in; Non-homologous-end-joining; Zebrafish