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Screen time for CRISPR CRISPR-Cas9 has been used to edit the genomes of organisms ranging from fruit flies to primates, but it has not been used in large-scale genetic screens in animals because generating, validating, and keeping track of large numbers of mutant animals is prohibitively laborious. Parvez et al . have developed Multiplexed Intermixed CRISPR Droplets, or MIC-Drop, a platform combining droplet microfluidics, en masse CRISPR injections, and barcoding, to enable large-scale genetic screens. In pilot phenotypic screens in zebrafish, MIC-Drop enabled rapid identification of the target of a small molecule and discovery of several new genes governing cardiovascular development. MIC-Drop is potentially scalable to thousands of targets and adaptable to diverse organisms and experiments. —DJ

Under conditions of inherent or induced mitochondrial dysfunction, cancer cells manifest overlapping metabolic phenotypes, suggesting that they may be targeted via a common approach. Here, we use multiple oxidative phosphorylation (OXPHOS)–competent and incompetent cancer cell pairs to demonstrate that treatment with α-ketoglutarate (aKG) esters elicits rapid death of OXPHOS-deficient cancer cells by elevating intracellular aKG concentrations, thereby sequestering nitrogen from aspartate through glutamic-oxaloacetic transaminase 1 (GOT1). Exhaustion of aspartate in these cells resulted in immediate depletion of adenylates, which plays a central role in mediating mTOR inactivation and inhibition of glycolysis. aKG esters also conferred cytotoxicity in a variety of cancer types if their cell respiration was obstructed by hypoxia or by chemical inhibition of the electron transport chain (ETC), both of which are known to increase aspartate and GOT1 dependencies. Furthermore, preclinical mouse studies suggested that cell-permeable aKG displays a good biosafety profile, eliminates aspartate only in OXPHOS-incompetent tumors, and prevents their growth and metastasis. This study reveals a novel cytotoxic mechanism for the metabolite aKG and identifies cell-permeable aKG, either by itself or in combination with ETC inhibitors, as a potential anticancer approach. Significance: These findings demonstrate that OXPHOS deficiency caused by either hypoxia or mutations, which can significantly increase cancer virulence, renders tumors sensitive to aKG esters by targeting their dependence upon GOT1 for aspartate synthesis.

G protein-coupled receptors (GPCRs) comprise the largest group of membrane receptors in eukaryotic genomes and collectively they regulate nearly all cellular processes. Despite the widely recognized importance of this class of proteins, many GPCRs remain understudied. G protein-coupled receptor 27 (Gpr27) is an orphan GPCR that displays high conservation during vertebrate evolution. Although, GPR27 is known to be expressed in tissues that regulate metabolism including the pancreas, skeletal muscle, and adipose tissue, its functions are poorly characterized. Therefore, to investigate the potential roles of Gpr27 in energy metabolism, we generated a whole body gpr27 knockout zebrafish line. Loss of gpr27 potentiated the elevation in glucose levels induced by pharmacological or nutritional perturbations. We next leveraged a mass spectrometry metabolite profiling platform to identify other potential metabolic functions of Gpr27. Notably, genetic deletion of gpr27 elevated medium-chain acylcarnitines, in particular C6-hexanoylcarnitine, C8-octanoylcarnitine, C9-nonanoylcarnitine, and C10-decanoylcarnitine, lipid species known to be associated with insulin resistance in humans. Concordantly, gpr27 deletion in zebrafish abrogated insulin-dependent Akt phosphorylation and glucose utilization. Finally, loss of gpr27 increased the expression of key enzymes in carnitine shuttle complex, in particular the homolog to the brain-specific isoform of CPT1C which functions as a hypothalamic energy senor. In summary, our findings shed light on the biochemical functions of Gpr27 by illuminating its role in lipid metabolism, insulin signaling, and glucose homeostasis.

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively. Prime editors are delivered as ribonucleoproteins to zebrafish embryos and human primary cells.

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 ( tfr2). TFR2 mutations in humans lead to HH Type 3, a rare but severe form of the disease. The tfr2 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.

Humans and many animals show 'freezing' behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and evaluated 10,000 compounds for their effects on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering, we identified the sigma-1 (σ1) receptor as having a role in the mechanism of behavioral switching and confirmed that known σ1 ligands also disrupt freezing behavior. Furthermore, mutation of the gene encoding σ1 prevented the behavioral effect of escape-inducing compounds. One compound, which we call finazine, potently bound mammalian σ1 and altered threat-response behavior in mice. Thus, pharmacological and genetic interrogation of the freezing response revealed σ1 as a mediator of threat responses in vertebrates.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

The transcription activator-like effector (TALE) nucleases, or TALENs, are customizable restriction enzymes that may be used to induce mutations at nearly any investigator-specified DNA sequence in zebrafish. The DNA-binding specificities of TALENs are determined by a protein array comprised of four types of TALE repeats, where each repeat recognizes a different DNA base. Here, we describe methods for constructing TALEN vectors that have been shown to achieve high success rates and mutation efficiencies in zebrafish. In addition, we discuss simple techniques and protocols that can be used to detect TALEN-induced mutations at almost any genomic locus. These methods should enable zebrafish researchers to quickly generate targeted mutations at their genes-of-interest.

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here, we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.

AML1-ETO is one of the most common chromosomal translocation products associated with acute myelogenous leukemia (AML). Patients carrying the AML1-ETO fusion gene exhibit an accumulation of granulocyte precursors in the bone marrow and the blood. Here, we describe a transgenic zebrafish line that enables inducible expression of the human AML1-ETO oncogene. Induced AML1-ETO expression in embryonic zebrafish causes a phenotype that recapitulates some aspects of human AML. Using this highly tractable model, we show that AML1-ETO redirects myeloerythroid progenitor cells that are developmentally programmed to adopt the erythroid cell fate into the granulocytic cell fate. This fate change is characterized by a loss of gata1 expression and an increase in pu.1expression in myeloerythroid progenitor cells. Moreover, we identify scl as an early and essential mediator of the effect of AML1-ETO on hematopoietic cell fate. AML1-ETO quickly shuts off scl expression,and restoration of scl expression rescues the effects of AML1-ETO on myeloerythroid progenitor cell fate. These results demonstrate that scl is an important mediator of the ability of AML1-ETO to reprogram hematopoietic cell fate decisions, suggesting that scl may be an important contributor to AML1-ETO-associated leukemia. In addition, treatment of AML1-ETO transgenic zebrafish embryos with a histone deacetylase inhibitor,Trichostatin A, restores scl and gata1 expression, and ameliorates the accumulation of granulocytic cells caused by AML1-ETO. Thus,this zebrafish model facilitates in vivo dissection of AML1-ETO-mediated signaling, and will enable large-scale chemical screens to identify suppressors of the in vivo effects of AML1-ETO.

Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.

Targeting the endothelial cell cycle as an antiangiogenic strategy has been difficult given the ubiquitous expression of critical cell cycle regulators. Here, we show that the antiangiogenic drug TNP-470 displays striking cell-type specificity insofar as it induces the expression of p21 CIP/WAF , a cyclin-dependent kinase inhibitor, in endothelial cells but not in embryonic or adult fibroblasts. Moreover, primary endothelial cells isolated from p53 −/− and p21 CIP/WAF−/− mice are resistant to the cytostatic activity of TNP-470. We also demonstrate that p21 CIP/WAF−/− mice are resistant to the antiangiogenic activity of TNP-470 in the basic fibroblast growth factor corneal micropocket angiogenesis assay. We conclude that TNP-470 induces p53 activation through a unique mechanism in endothelial cells leading to p21 CIP/WAF expression and subsequent growth arrest.

Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light but does not require the eyes, pineal gland, or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical nonvisual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Usingin vivocalcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB , account for ∼24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ∼50%. We hypothesize that the interaction between RUNX1 and CBFβ is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1–CBFβ interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB -dependent transactivation in reporter assays, and repress runx1 -dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1–ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB–MYH11 leukemia model. Our data thus confirmed that RUNX1–CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

The number of possible small organic molecules of different structure is virtually limitless. One of the main goals of chemical biologists is to identify, from this “chemical space”, entities that affect biological processes or systems in a specific manner. This can lead to a better understanding of the regulation and components of various biological machineries, as well as provide insights into efficacious therapeutic targets and drug candidates. However, the challenges confronting chemical biologists are multiple. How do we efficiently identify compounds that possess desirable activities without unwanted off-target effects? Once a candidate compound has been found, how do we determine its mode of action? In this Prospects piece, we call attention to recent studies using embryonic and larval zebrafish to illustrate the breadth and depth of questions in chemical biology that may be addressed using this model, and hope that they can serve as catalysts for future investigational ideas.

Embryonic zebrafish have long been used for lineage-tracing studies. In zebrafish embryos, the cell fate identities can be determined by whole-mount in situ hybridization, or by visualization of live embryos if using fluorescent reporter lines. We use embryonic zebrafish to study the effects of a leukemic oncogene AML1-ETO on modulating hematopoietic cell fate. Induced expression of AML1-ETO is able to efficiently reprogram hematopoietic progenitor cells from erythroid to myeloid cell fate. Using the zebrafish model of AML1-ETO, we performed a chemical screen to identify small molecules that suppress the cell fate switch in the presence of AML1-ETO. The methods discussed herein may be broadly applicable for identifying small molecules that modulate other cell fate decisions.

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 the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. Conclusions and Significance In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months.

It has been proposed that inhibitors of an oncogene's effects on multipotent hematopoietic progenitor cell differentiation may change the properties of the leukemic stem cells and complement the clinical use of cytotoxic drugs. Using zebrafish, we developed a robust in vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO. Screening for modifiers of AML1-ETO-mediated hematopoietic dysregulation uncovered unexpected roles of COX-2- and beta-catenin-dependent pathways in AML1-ETO function. This approach may open doors for developing therapeutics targeting oncogene function within leukemic stem cells.

Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A-P) patterning to identify the target of DTAB, a compound that caused A-P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors gamma and beta. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene ( MAP2 ) resulted in an early gastrulation defect, which is bypassed in double MetAP-2/p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.

Recent advances in cell and molecular biology have generated important tools to probe developmental questions. In addition, new genetic model systems such as Danio rerio now make large-scale vertebrate early developmental mutant screens feasible. Nonetheless, some developmental questions remain difficult to study because of the need for finer temporal, spatial, or tuneable control of gene function within a developmental system. New uses for old teratogens as well as novel chemical modulators of development have begun to fill this void.

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.