RESUMO
Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.
Assuntos
Baculoviridae , Sistemas CRISPR-Cas , Edição de Genes , Vetores Genéticos , Baculoviridae/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Animais , Células HEK293RESUMO
Genome editing technologies, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas in particular, have revolutionized the field of genetic engineering, providing promising avenues for treating various genetic diseases. Chronic kidney disease (CKD), a significant health concern affecting millions of individuals worldwide, can arise from either monogenic or polygenic mutations. With recent advancements in genomic sequencing, valuable insights into disease-causing mutations can be obtained, allowing for the development of new treatments for these genetic disorders. CRISPR-based treatments have emerged as potential therapies, especially for monogenic diseases, offering the ability to correct mutations and eliminate disease phenotypes. Innovations in genome editing have led to enhanced efficiency, specificity and ease of use, surpassing earlier editing tools such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs). Two prominent advancements in CRISPR-based gene editing are prime editing and base editing. Prime editing allows precise and efficient genome modifications without inducing double-stranded DNA breaks (DSBs), while base editing enables targeted changes to individual nucleotides in both RNA and DNA, promising disease correction in the absence of DSBs. These technologies have the potential to treat genetic kidney diseases through specific correction of disease-causing mutations, such as somatic mutations in PKD1 and PKD2 for polycystic kidney disease; NPHS1, NPHS2 and TRPC6 for focal segmental glomerulosclerosis; COL4A3, COL4A4 and COL4A5 for Alport syndrome; SLC3A1 and SLC7A9 for cystinuria and even VHL for renal cell carcinoma. Apart from editing the DNA sequence, CRISPR-mediated epigenome editing offers a cost-effective method for targeted treatment providing new avenues for therapeutic development, given that epigenetic modifications are associated with the development of various kidney disorders. However, there are challenges to overcome, including developing efficient delivery methods, improving safety and reducing off-target effects. Efforts to improve CRISPR-Cas technologies involve optimizing delivery vectors, employing viral and non-viral approaches and minimizing immunogenicity. With research in animal models providing promising results in rescuing the expression of wild-type podocin in mouse models of nephrotic syndrome and successful clinical trials in the early stages of various disorders, including cancer immunotherapy, there is hope for successful translation of genome editing to kidney diseases.
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Triple-negative breast cancer (TNBC) is characterised by its aggressiveness and resistance to chemotherapy, demanding the development of effective strategies against its unique characteristics. Derived from lapacho tree bark, ß-lapachone (ß-LP) selectively targets cancer cells with elevated levels of the detoxifying enzyme NQO1. Hydroxytyrosol (HT) is a phenolic compound derived from olive trees with important anticancer properties that include the inhibition of cancer stem cells (CSCs) and metastatic features in TNBC, as well as relevant antioxidant activities by mechanisms such as the induction of NQO1. We aimed to study whether these compounds could have synergistic anticancer activity in TNBC cells and the possible role of NQO1. For this pourpose, we assessed the impact of ß-LP (0.5 or 1.5⯵M) and HT (50 and 100⯵M) on five TNBC cell lines. We demonstrated that the combination of ß-LP and HT exhibits anti-proliferative, pro-apoptotic, and cell cycle arrest effects in several TNBC cells, including docetaxel-resistant TNBC cells. Additionally, it effectively inhibits the self-renewal and clonogenicity of CSCs, modifying their aggressive phenotype. However, the notable impact of the ß-LP-HT combination does not appear to be solely associated with the levels of the NQO1 protein and ROS. RNA-Seq analysis revealed that the combination's anticancer activity is linked to a strong induction of endoplasmic reticulum stress and apoptosis through the unfolded protein response. In conclusion, in this study, we demonstrated how the combination of ß-LP and HT could offer an affordable, safe, and effective approach against TNBC.
Assuntos
Apoptose , Proliferação de Células , NAD(P)H Desidrogenase (Quinona) , Naftoquinonas , Álcool Feniletílico , Álcool Feniletílico/análogos & derivados , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Naftoquinonas/farmacologia , Linhagem Celular Tumoral , Álcool Feniletílico/farmacologia , Apoptose/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Sinergismo Farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacosRESUMO
CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.
Assuntos
Baculoviridae , Sistemas CRISPR-Cas , DNA , Edição de Genes , Proteínas Virais , Animais , Humanos , Baculoviridae/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/métodos , Proteínas Virais/genética , Linhagem CelularRESUMO
Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.
Assuntos
Condução de Veículo , Endoderma , Proteína 1 Semelhante ao Fator 7 de Transcrição , Animais , Feminino , Camundongos , Gravidez , Blastocisto , Diferenciação Celular , Camadas GerminativasRESUMO
CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic ß-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.
Assuntos
Baculoviridae , Sistemas CRISPR-Cas , Baculoviridae/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes , Vetores Genéticos , HumanosRESUMO
The baculovirus expression vector system (BEVS) delivers high yield heterologous protein expression and is widely used in academic and industrial R&D. The proteins produced enable many applications including structure/function analysis, drug screening and manufacture of protein therapeutics. Vital cellular functions are controlled by multi-protein complexes, MultiBac, a BEVS specifically designed for heterologous multigene delivery and expression, has unlocked many of these machines to atomic resolution studies. Baculovirus can accommodate very large foreign DNA cargo for faithful delivery into a target host cell, tissue or organism. Engineered MultiBac variants exploit this valuable feature for delivery of customized multifunctional DNA circuitry in mammalian cells and for production of virus-like particles for vaccines manufacture. Here, latest developments and applications of the MultiBac system are reviewed.
Assuntos
Baculoviridae , Proteínas , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , DNA/genética , DNA/metabolismo , Vetores Genéticos/genética , Mamíferos/genética , Proteínas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The Wnt/ß-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel ß-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as ß-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a ß-catenin-dependent manner. Finally, ß-catenin (ΔC) isoforms can rescue ß-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Via de Sinalização Wnt , beta Catenina/metabolismo , Alelos , Animais , Biomarcadores/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Autorrenovação Celular/genética , Células Cultivadas , Ectoderma/metabolismo , Endoderma/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Isoformas de Proteínas/metabolismo , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
DNA delivery is at the forefront of current research efforts in gene therapy and synthetic biology. Viral vectors have traditionally dominated the field; however, nonviral delivery systems are increasingly gaining traction. Baculoviruses are arthropod-specific viruses that can be easily engineered and repurposed to accommodate and deliver large sequences of exogenous DNA into mammalian cells, tissues, or ultimately organisms. These synthetic virus-derived nanosystems (SVNs) are safe, readily customized, and can be manufactured at scale. By implementing clustered regularly interspaced palindromic repeats (CRISPR) associated protein (CRISPR/Cas) modalities into this system, we developed SVNs capable of inserting complex DNAs into genomes, at base pair precision. We anticipate a major role for SVNs as an attractive alternative to viral vectors in accelerating genome engineering and gene therapy applications in the future.
RESUMO
Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Trimetoprima/farmacologia , Animais , Anti-Infecciosos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Microscopia Confocal , Proteína Vermelha FluorescenteRESUMO
Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.
Assuntos
Diferenciação Celular , Proliferação de Células , Epigênese Genética , Homeostase , Células-Tronco Embrionárias Murinas/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Metilação de DNA , Camundongos , Células-Tronco Embrionárias Murinas/citologiaRESUMO
Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with the desired features to probe cell response are still lacking. Here, combining tools from Control Engineering and Synthetic Biology (cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signaling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual in silico counterparts. Ultimately, this platform can be used to probe gene networks and signaling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.
Assuntos
Expressão Gênica , Microfluídica/métodos , Transdução de Sinais , Biologia Sintética/métodos , Animais , Automação , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.
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Sistemas CRISPR-Cas/genética , Genes/genética , Vetores Genéticos/genética , Medições Luminescentes/métodos , Imagem com Lapso de Tempo/métodos , Animais , Linhagem Celular , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Hibridização in Situ Fluorescente/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Reprodutibilidade dos Testes , Telômero/genética , Telômero/metabolismoRESUMO
Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.
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Reprogramação Celular/genética , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Animais , Autorrenovação Celular/genética , Imunofluorescência , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/ß-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/ß-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of ß-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.
Assuntos
Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Células-Tronco Embrionárias Murinas/metabolismo , Via de Sinalização Wnt/genética , Animais , Sequência de Bases , Western Blotting , Proliferação de Células/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Homotypic and heterotypic cell-to-cell fusion are key processes during development and tissue regeneration. Nevertheless, aberrant cell fusion can contribute to tumour initiation and metastasis. Additionally, a form of cell-in-cell structure called entosis has been observed in several human tumours. Here we investigate cell-to-cell interaction between mouse mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs). MSCs represent an important source of adult stem cells since they have great potential for regenerative medicine, even though they are also involved in cancer progression. We report that MSCs can either fuse forming heterokaryons, or be invaded by ESCs through entosis. While entosis-derived hybrids never share their genomes and induce degradation of the target cell, fusion-derived hybrids can convert into synkaryons. Importantly we show that hetero-to-synkaryon transition occurs through cell division and not by nuclear membrane fusion. Additionally, we also observe that the ROCK-actin/myosin pathway is required for both fusion and entosis in ESCs but only for entosis in MSCs. Overall, we show that MSCs can undergo fusion or entosis in culture by generating distinct functional cellular entities. These two processes are profoundly different and their outcomes should be considered given the beneficial or possible detrimental effects of MSC-based therapeutic applications.
Assuntos
Células Híbridas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Embrionárias Murinas/citologia , Actinas/metabolismo , Animais , Fusão Celular , Técnicas de Cocultura , Entose , Células Híbridas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Miosinas/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Cyclic activation of the Wnt/ß-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ß-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/ß-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/ß-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/ß-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.