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

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

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

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

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

Efficiency of the tested CompoZr and CoDA ZFNs.

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

An EvoPrint view of a genomic segment of human DNA selected and then screened for mutations.

<p>(A) A multiple species comparison was performed where bases conserved in all but one of the test species, relaxed EvoPrint, appear as black uppercase letters (these alignments were among Human, Marmoset, Chimpanzee, Rhesus-Monkey, Horse, Platypus and Opossum) and are displayed in context with non-conserved bases (lower case, grey) spanning the entire 804 bp element shown in blue. (B) Analysis of the repetitive and palindromic structure of the EvoPrint using <i>cis</i>-Decoder identifies further substructure of its conserved sequence blocks (CSB). Distinct elements (>6 bp) are highlighted in yellow. These CSC analyzed and presented are contained within the element in common [>50% conservation between human and zebrafish, outlined in red] where most variants were detected.</p

Unique Alterations of an Ultraconserved Non-Coding Element in the 3′UTR of <em>ZIC2</em> in Holoprosencephaly

<div><p>Coding region alterations of <em>ZIC2</em> are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human <em>ZIC2</em>. We demonstrate that an 804 bp element in the 3′ untranslated region (3′UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are <em>de novo</em>, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.</p> </div

<i>ZIC2</i> enhancer mutational screening and control results by TaqMan Assay.

a<p><i>De novo</i>, parental testing confirms biological relatedness of parental DNA; ^b Variant allele <i>in cis</i> with a <i>ZIC2</i> c.1215dupC (p.Ser406Glnfs*91) based on co-amplification and subcloning; ^c Subject LCL7897 is the affected sibling of proband LCL301 (both are carriers of a SHH p.Cys24* mutation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039026#pone.0039026.s005" target="_blank">Table S1</a>). ^d Described as <i>de novo</i> based on normal sequence of both parents (done in Brazil). ^e Proband also has novel mutations in <i>TGIF</i> (c.289A>G, p.Met97Val). SNPs are highlighted in bold.</p