Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through th

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications

Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

DTAB affects the expression of early developmental markers.

<p><i>In situ</i> hybridizations of zebrafish embryos show that treatment with DTAB results in severe reduction of <i>krox20</i>, <i>pax2a</i>, <i>myoD</i>, <i>ntl</i>, <i>gsc</i>, and <i>raldh2</i> staining. DTAB treated embryos exhibited an upregulation of <i>cyp26a1</i> transcripts in the tail and head. Embryos were treated with 12.5 µM DTAB or DMSO control from 4hpf onwards until fixation. Embryos were fixed at 75% epiboly (G–L), 10-somite (M–N), 18-somite (C–F) and 26-somite (A,B) stages. A–B, E–F, M–N are lateral views with anterior to the left and dorsal to the top. C–D are dorsal views with anterior to the left. G–L are dorsal views with the animal pole to the top.</p

DTAB causes anterior-posterior axis defects.

<p>a) Chemical structure of DTAB. b) Embryos treated with higher concentrations of DTAB showed more anterior-posterior patterning defects than those treated with lower concentrations. Zebrafish embryos were treated from 1hpf to 48hpf with different concentrations of DTAB. c) Embryos treated earlier in development, and thus for longer duration were more severely affected than those treated later. Embryos were exposed to 3 µM DTAB from different times of development to 48hpf and compared to embryos treated with the vehicle DMSO. Vehicle (DMSO) treated controls developed normally. All observations were made at 48hpf.</p

Zebrafish small-molecule screen identifies compounds that effect development.

<p>a) 4% of the 5760 compounds screened affected zebrafish embryos. 19% of the active compounds caused lethality. 175 compounds with detectable effects on zebrafish development were identified. b–c) The small molecules used for screening had molecular weights ranging from approx. 200 daltons to 700 daltons and had log P (partition coefficient) values ranging from −10 to +15. However, the log P of the bioactive compounds were restricted to 0 to +12. b) log P values between octanol and water of all the compounds screened plotted against their molecular weights. c) log P values of all the bioactive compounds (excluding the toxic compounds) plotted against their molecular weights.</p

Small Molecule Inhibitors of NFkB Reverse Iron Overload and Hepcidin Deregulation in a Zebrafish Model for Hereditary Hemochromatosis Type 3

Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasian populations, with no viable therapeutic options except phlebotomy. We describe a zebrafish model of human HH (HH) created by targeted mutagenesis of the gene encoding transferrin receptor 2 (<i>tfr2</i>). <i>TFR2</i> mutations in humans lead to HH Type 3, a rare but severe form of the disease. The <i>tfr2</i> mutant model in zebrafish recapitulates the defining features of HH3: iron overload and suppression of hepcidin, the iron regulatory hormone. Using in vivo chemical screens in zebrafish embryos, we identify a new small molecule inducer of hepcidin: SC-514, a specific chemical inhibitor of NFkB signaling. Using independent small molecule inhibitors of the NFkB pathway, we demonstrate that inhibition of NFkB signaling causes induction of hepcidin transcription and reduction of iron overload in the HH3 model. This first successful chemical intervention for hereditary hemochromatosis may also have relevance in treatment of other very prevalent iron regulatory iron overload disorders such as thalassemia