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

<div><p>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.</p> </div

List of all mutations identified by founder screening and genotyping of F1 adults.

<p>For each target gene, the total number of mutations, total number of germline-transmitting founders, and number of mutations transmitted by each founder are given. For example, for <i>cbfb</i> 4×1, 1×2, 1×3 indicates 4 founders each transmitted a single mutation, one founder transmitted two mutations and another founder transmitted three mutations, for a total of 9 mutations. In all cases, the Wild-type sequences with spacer marked in red are shown at the top followed by the sequences of the mutant alleles. Deletions are marked by red dashes highlighted in yellow and insertions by lower case letters highlighted in blue color. The nature of each mutation is indicated on the right side of the sequence, Δ indicates deletion, + indicates insertion. In some cases, identical mutations were transmitted by multiple founders and these are indicated by x # Fo (meaning times # of founders). Finally, additional mutations whose sequences were not determined are listed at the bottom for each gene.</p

Fluorescent PCR strategy and examples of mutant peaks from founder screening and F1 genotyping.

<p>A) Schematic of the fluorescent PCR strategy. Target region is shown as two black lines. Forward primers are denoted as FAM (blue star) -labeled M13F primer (M13F-FAM) and gene-specific primer with M13F tail (M13F-tailed forward primer). Reverse primer is denoted as the gene-specific part and the PIG-tail sequence (PIG-tailed reverse primer). B and C) Founder screening for <i>ak2</i> using 4 pooled embryos per well (B) and genotyping of heterozygous adults from F1 progeny of the corresponding transmitting founders (C). Fragment size scales are shown on the top and each fragment's size is marked underneath the peak. Vertical scale marks the intensity of the peaks. Black arrows mark the 257 bp peak corresponding to the Wild-type allele observed in all samples. The top panel in B is a Wild-type control DNA sample, the middle panel is a founder transmitting a 1 bp insertion mutation (258 bp, marked by a red arrow) and the bottom panel shows a founder transmitting two mutations, a 13 bp deletion (244 bp, marked by a green arrow) and a 4 bp insertion (261 bp, marked by a blue arrow). In C each panel shows a heterozygous adult zebrafish and color-matched arrows mark the mutant peaks.</p

Flowchart of step-by-step experimental procedures for generating mutant zebrafish lines with ZFNs.

<p>Steps involving ZFN design, mRNA injections, and toxicity assessment are shown in blue color, efficiency testing using somatic lesion analysis in green color and founder screening steps in orange color.</p

Rate of germline transmission of ZFN-induced mutations.

a<p>: Counted only the founders where progeny was analyzed by both methods.</p>b<p>: Calculated assuming only one embryo in the pools with mutant peaks is heterozygous.</p

Target sequences and in vitro activity of CompoZr ZFNs.

a<p>: Spacer sequences are shown in lower case letters in italics. Bold letters denote polymorphic sites. Underlined letters denote the splice site. Lower case letters denote nucleotides flanking the left and right ZFN recognition sequences and gaps between adjacent ZFPs.</p>b<p>: <i>in vitro</i> activity was measured by Mel1 reporter assay and required at >50% except for <i>cbfb</i> where zebrafish SJD.1 cells were transfected and activity was measured by surveyor assay (>1% required).</p

Efficiency of the tested CompoZr and CoDA ZFNs.

<p>Efficiency of the tested CompoZr and CoDA ZFNs.</p

Clinical ocular phenotype in C57BL/6-<i>Pax2^+/A220G</i> mice compared to wild-type, C57BL/6 mice.

<p>(A) Fundus photograph of C57BL/6 mouse showing normal optic nerve and radial pattern of retinal blood vessels. (B) Fundus photograph of C57BL/6-<i>Pax2^+/A220G</i> mouse showing congenital excavation of the optic nerve head with peripapillary pigment changes (arrow). (C) Lectin immunofluorescence of wild-type C57BL/6 mouse showing normal, radial vessel patterning. (D,E) Lectin immunofluorescence of C57BL/6-<i>Pax2^+/A220G</i> mice showing abnormal vascular patterning, including curving of vessels towards the dorsal retina (D, arrows, d = dorsal, v = ventral) and separation of the central retinal vascular trunks (E, arrows). Histologic section of a <i>Pax2^+/+</i> (F) and a <i>Pax2^+/A220G</i> (G) mouse eye through the optic nerve and peripapillary retina showing abnormal excavation of the optic nerve (G, arrow) and retinal rosette formation (G, arrowhead). Remnants of the <i>tunica vasculosis lentis</i> and mild extension of the retinal pigment epithelium were variably noted in histopathology from other <i>Pax2^A220G/+</i> eyes (data not shown).</p

Threshold cycle (C_t) quantification of real-time, reverse-transcriptase PCR of wild-type and mutant <i>Pax2</i> transfected NIH/3T3 cells shows no significant difference in steady-state levels of <i>Pax2</i> mRNA relative to Gapdh.

<p>Threshold cycle (C_t) quantification of real-time, reverse-transcriptase PCR of wild-type and mutant <i>Pax2</i> transfected NIH/3T3 cells shows no significant difference in steady-state levels of <i>Pax2</i> mRNA relative to Gapdh.</p

Comparison of wild-type and mutant Pax2 protein transactivation and expression in cell culture.

<p>NIH/3T3 cells were transfected with expression constructs for wild-type or mutant <i>Pax2</i> along with a <i>Pax2</i>-responsive luciferase reporter gene. All three mutants tested show reduced ability to transactivate (A). When steady-state levels of Pax2 protein were compared on Western blots from these experiments, mutants showed consistently lower levels of expression (B). Similar findings were observed when these experiments were replicated in COS-7 cells (data not shown).</p