Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs

Abstract

Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich) or the CoDA (context-dependent assembly) platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1) evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2) a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5%) when compared to CopmoZr ZFNs (9–98%). This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

“…Previously, we targeted 26 genes with ZFNs or TALENs and successfully generated multiple knockout alleles for 21 genes (Sood et al 2013). In addition to the substantial reduction in cost and increased ease of design and assembly for CRISPR targets compared to ZFNs and TALENs, our data showed that the CRISPRs were significantly more efficient in generating genetic mutations (Fig.…”

“…Injected fish were grown to adulthood and screened for germline transmission of CRISPR-induced mutations by fluorescence PCR (Sood et al 2013). Each putative founder fish was crossed with a wild-type fish and embryos were harvested at 24-72 h post-fertilization in 96-well plates, one embryo per well.…”

CBFβ and RUNX1 are required at 2 different steps during the development of hematopoietic stem cells in zebrafish

“…Injection of mRNA, founder screening, and identification of cbfb heterozygous adult fish has been described in detail previously. 30 For each experiment, cbfb-Del4 and cbfb-Ins4 were genotyped by fluorescent polymerase chain reaction (PCR) using a mixture of M13F-tailed (59-TGTAAAACGACGGCCAGT-39) cbfb-specific forward primer (59-ATGCTCGGGCCTGGCTTTCT-39), 6-FAM-labeled M13F primer, and PIG-tailed (59-GTGTCTT-39) cbfb-specific reverse primer (59-AGG GGCGTGAGTTAGAGT-39) in the PCR mix. 30 Genotyping of fixed samples has been performed as above but using a different PCR mix (Sigma RED Extract-N-Amp PCR Ready Mix R4775 REDExtract-N-Amp PCR Ready Mix, R4775; Sigma-Aldrich).…”

“…32,33 The selected CompoZr ZFN pair targeted a specific region within cbfb exon 3 ( Figure 1A). 30 Among 9 mutations identified 30 from 6 germline-transmitting founders, we selected 2 mutations predicted to cause frameshifts with premature terminations, cbfb hg10 (c.215delACCT, p.N72IfsX25) and cbfb hg11 (c.215insACCT, p.H74PfsX43), denoted here as cbfb del4 and cbfb ins4 ( Figure 1B). In order to test whether the mutations lead to loss of cbfb expression, we evaluated the presence of cbfb transcripts in cbfb del4/del4…”

Methods for targeted mutagenesis in zebrafish using TALENs

“…To date, three major types of customizable endonucleases have been developed, namely zinc finger nucleases (ZFNs) [5–13], transcription activator-like effector nucleases (TALENs) [10,14–21] and clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided Cas9 (CRISPR/Cas) nucleases [22–32]. All three classes have been shown to cause efficient target gene disruption in zebrafish [5,6,8,9,14,17,19,27,33–39]. Nevertheless, each platform differs in the ease of their construction methods, potential off-target activities and the theoretical targeting range.…”