Urp1 and Urp2 act redundantly to maintain spine shape in zebrafish larvae.

Urp1 and Urp2 are two neuropeptides, members of the Urotensin 2 family, that have been recently involved in the control of body axis morphogenesis in zebrafish. They are produced by a population of sensory spinal neurons, called cerebrospinal fluid contacting neurons (CSF-cNs), under the control of signals relying on the Reissner fiber, an extracellular thread bathing in the CSF. Here, we have investigated further the function of Urp1 and Urp2 (Urp1/2) in body axis formation and maintenance. We showed that urp1;urp2 double mutants develop strong body axis defects during larval growth, revealing the redundancy between the two neuropeptides. These defects were similar to those previously reported in uts2r3 mutants. We observed that this phenotype is not associated with congenital defects in vertebrae formation, but by using specific inhibitors, we found that, at least in the embryo, the action of Urp1/2 signaling depends on myosin II contraction. Finally, we provide evidence that while the Urp1/2 signaling is functioning during larval growth, it is dispensable for embryonic development. Taken together, our results show that Urp1/2 signaling is required in larvae to promote correct vertebral body axis, most likely by regulating muscle tone.

Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling ß-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle ß-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous ß-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.

De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells.

BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.

Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Cancer translocations in human cells induced by zinc finger and TALE nucleases.

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

Gene targeting in rats using transcription activator-like effector nucleases.

The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Tetramolecular quadruplex stability and assembly.

Guanine quadruplexes (G4) are unusual four-stranded nucleic acid structures formed by G-rich DNA/RNA. Beyond their likely biological relevance, the self-assembly, stability, and rigidity of these structures are also interesting for nanotechnology and biotechnology applications. Therefore, efforts are carried out to understand the rules that govern stability and folding of G-quadruplexes. We focus this chapter on tetramolecular conformations which are simple tractable models. We report here the experimental parameters, molecules, and modifications that affect thermal stability and/or association kinetics of these structures. Some chemical modifications which facilitate tetramolecular quadruplex formation and can be useful for nano- or biotechnology are also described.