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.
Highlights
The successful completion of the Human Genome Project and the advances in sequencing technologies in the past decade have led to the sequencing of complete genomes of many species
Recent advances in targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have overcome the limitations of previously available reverse genetics techniques in zebrafish that relied on random mutagenesis with chemical or retroviral methods
It would be of great benefit to develop general guidelines for successful application of ZFNs and TALENs to large-scale zebrafish knockout projects
Summary
The successful completion of the Human Genome Project and the advances in sequencing technologies in the past decade have led to the sequencing of complete genomes of many species. Sequencing of whole genomes and exomes has replaced traditional positional cloning approaches for the identification of disease genes and cancer-causing mutations. Sifting through the large number of variants detected through whole genome and exome sequencing to determine the variants responsible for the phenotype under investigation is limited by our knowledge of the function of all protein coding genes. Generation of genetic mutants relied on chemical and insertional mutagenesis approaches that caused random mutations throughout the genome [5,6]. Chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) followed by forward (phenotype-based) and reverse (gene-based, termed TILLING for Targeting Induced Local Lesions IN Genomes) screens became popular among the zebrafish community [7,8,9,10]. Each founder fish typically carries hundreds of ENU-induced mutations, which may slow and/or complicate phenotype analysis of the mutant fish
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