Gene editing technologies that make use of homology-dependent recombination after digestion by meganucleases, such as CRISPRs (clustered regularly interspaced short palindromic repeats)1 or TALENs (transcription activator-like effector nucleases),2 today enable us to accurately modify in vivo any genomic sequence of interest in the zebrafish. Like in classical transgenic methods, the identification of rare G1 individuals carrying an edited allele usually relies on reporter gene expression, such as a fluorescent protein. However, the inclusion of a reporter gene may not be appropriate in the case of precise editing events, such as site-directed mutagenesis or protein-tag addition. In addition, although recent improvements increased efficiency of genome editing protocols,3 carrier fish usually comprise a small proportion of G0 individuals (in our hands, we routinely observed from 5% to 15% G0 carriers). To circumvent these limitations, we describe here a simple, efficient, and rapid method of identifying G0 males with mosaic germ line cells and heterozygous G1 individuals (Fig. 1).
Schematic representation of the screening protocol. After injection at the one-cell stage, G0 individuals were raised to adulthood, and males carrying mosaic germ line cells were identified by PCR analysis of sperm DNA (A). Selected carrier males were crossed individually to wild-type AB females. For each clutch, DNA was extracted from several batches of embryos (four or five embryos per batch), collectively representing approximately 20%–25% of the clutch, and underwent PCR analysis (B). The remaining embryos from clutches with positive PCR results were raised to adulthood, and editing events were identified in G1 individuals using PCR analysis of tail-clip DNA (C). CRISPRs, clustered regularly interspaced short palindromic repeats; PCR, polymerase chain reaction; TALEN, transcription activator-like effector nuclease.
This efficient method of identifying G0 males with mosaic germ line cells should be helpful for the fast generation of models in zebrafish. Although polymerase chain reaction (PCR) screening is not appropriate to detect editing events encompassing less than three nucleotides or deletions, the described method is time saving for most applications involving the introduction of foreign DNA at a specific genomic site (genome knock-in).
Concise Workflow
1. Sperm recovery: G0 male zebrafish were anesthetized in tricaine solution and immediately transferred to a Petri dish containing fresh E3 medium. The fish were maintained under a dissecting microscope, and gentle finger pressure was applied to the abdomen in a head-to-tail direction: the expelled sperm was recovered with a micropipette in a total volume of 15 to 30 μL of E3 medium.
2. Sperm DNA extraction: One hundred microliters of extraction buffer (10 mM Tris, 200 mM NaCl, 10 mM EDTA, 0.5% SDS, and 200 mg/mL of Proteinase K) was added to the sperm extract, which was then incubated for 1 h at 55°C.
3. PCR detection of editing events: One microliter of the crude DNA sperm extract was used for direct PCR using Terra PCR Direct kit (Clontech).
Footnotes
Disclosure Statement
No competing financial interests exist.
References
1.
AuerTO, DuroureK, De CianA, ConcordetJP, Del BeneF. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res, 2014; 24:142–153.
2.
ZuY, TongX, WangZ, LiuD, PanR, LiZ, et al.TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods, 2013; 10:329–331.
3.
HoshijimaK, JurynecMJ, GrunwaldDJ. Precise editing of the zebrafish genome made simple and efficient. Dev Cell, 2016; 36:654–667.
