11 research outputs found
CRISPR-Cas9 to model retinitis pigmentosa caused by mutations in splicing factors in C. Elegans
[eng] Retinitis pigmentosa (RP) is a rare, heterogenic, and hereditary disease that produces gradual loss of the visual field and can cause blindness. Mutations causing RP are still unknown in about 50% of the cases. By CRISPR-Cas9, we mimicked a few splicing-related RP mutations (s-adRP) in PRPF8/prp-8 and SRNPN200/snpr-200 that were used for drug screens, identify potential disease modifiers, investigate mechanisms of the disease, and work on a system to provide functional information for gene variants. One of the alleles generated, displaying an overt phenotype, was used in a small-scale drug screen to identify small molecules capable of alleviating the phenotype. Unexpectedly, we found an FDA-approved drug having a detrimental effect on some of the s-adRP mutant strains. Since RP onset and progression are highly variable due to environmental or genetic modifiers, C. elegans could help RP prognosis by identifying such modifiers. We performed a small-scale RNAi screen on RP mutants with no overt phenotypes and found genetic interactions with other splicing- related genes: isy-1/ISY1, mog-2/SNRPA1, and cyn-15/PPWD1. Thus, secondary mutations in these genetic interactors could act as modifiers of the course of the disease. The mechanism by which s-adRP mutations selectively cause retinal deterioration is unknown. We detected some hints of genome instability in s-adRP mutants, which might explain the degenerative nature of the disease. We are taking steps towards establishing C. elegans as an RP diagnosis model by evaluating the functional impact of potential RP mutations, or variants of uncertain significance (VUS), in worms. For that purpose, we set a panel of features associated with s-adRP mutations, including a genetic interaction with a CRISPR-edited Slow Polymerase II mutant (ama-1(cer135[R743H])), mortal germline, or aberrant splicing events at specific transcripts. We partially humanized the sequence encoding the splicing factors prp-3 in the endogenous locus to investigate if such humanization is beneficial for functional studies of VUS. Therefore, our RP research line demonstrates the value of C. elegans for investigating rare diseases and for providing valuable information in search of drugs, diagnosis, and prognosis
Arrhythmic Effects Evaluated on <i>Caenorhabditis elegans</i>: The Case of Polypyrrole Nanoparticles
Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs
Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin
Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.Instituto de Salud Carlos III PI15/00895 PI16/01898European Regional Development Fund/FEDERNetherlands Organization for Scientific Research 711.014.005Sociedad Española de Oncología MédicaMinisterio de Economía y Competitividad BFU2007-67123 BFU2015-64408-PEuropean Social Fund BFU2015-64408-
Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin
Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the proapoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1. This article has an associated First Person interview with the first author of the paper
Fluorizoline-induced apoptosis requires prohibitins in nematodes and human cells
We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.This work was supported by grants from the Agencia Estatal de Investigación (Ministerio de Ciencia e Innovación), European Regional Development Fund (ERDF), the European Research Council, the Junta de Andalucía and the Instituto de Salud Carlos III (ISCIII) (SAF2017-83178-R to J.G.; PID2019-107991RB-I00 to R.L.; ERC-2011-StG-281691 and C2A ID: 42571/Exp: 70806 to M.A-S; PI15-00895 to J.C.). J.S-E and I.S-V are recipients of research fellowships from the Ministerio de Ciencia e Innovación. S.N-V is recipient of a research fellowship from Universitat de Barcelona. MD.M-B was supported by the Plan de Empleo Juvenil (EJP09) from the Junta de Anadalucía. D.K has a FI AGAUR fellowship from Generalitat de Catalunya
Synthetic biology in multicellular organisms: Opportunities in nematodes
Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.ISSN:0006-3592ISSN:1097-029
Arrhythmic Effects Evaluated on Caenorhabditis elegans: The Case of Polypyrrole Nanoparticles
Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes’ pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs’ biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs.This work was supported by the Ministry of Science and Innovation (MICINN) “Severo Ochoa” program for centers of excellence through the BEAT project (FUNFUTURE-FIP-2020), MICINN project PID2021-122239OB-I00 and partly supported by the European Union’s Horizon Europe research and innovation program under grant agreement No 101057527 (NextGEM). Funded by the European Union. Some experiments were performed at MIRAS beamline at ALBA Synchrotron with the collaboration of ALBA staff. SYS is enrolled in the Materials Science Ph.D. program of Universitat Autonoma de Barcelona (UAB). SYS acknowledges financial support from DOC-FAM, European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 754397. AL and SYS participate in the networks of EPNOE, Red Nanocare2.0 , and the CSIC Interdisciplinary Platform for Sustainable Plastics toward a Circular Economy, SUSPLAST. The authors acknowledge the use of Biorender.comPeer reviewe
Genetic and cellular sensitivity of to the chemotherapeutic agent cisplatin
Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.This article has an associated First Person interview with the first author of the paper
Ancestral function of Inhibitors-of-kappaB regulates Caenorhabditis elegans development
Mammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs in Caenorhabditis elegans, which lacks NF-κB nuclear effectors. We found that nfki-1 and ikb-1 mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role for C. elegans IκBs in chromatin regulation. Further supporting this possibility (1) we detected NFKI-1 in the nucleus of cells; (2) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (3) and associate with chromatin in vivo, and (4) mutations in nfki-1 and ikb-1 alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development
Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
PAM requirement is a constraint for genome editing but this has been circumvented by engineered Cas9 nucleases as SpG and SpRY recognizing minimal PAM sequences. Here, the authors validate and optimize SpG and SpRY in vivo expanding the targeting landscape in animals. The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals