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1.
Nat Biotechnol ; 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38297187

RESUMO

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.

2.
Mol Ther ; 31(7): 2266-2285, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-36934299

RESUMO

The human T cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that persists as a provirus in the genome of infected cells and can lead to adult T cell leukemia (ATL). Worldwide, more than 10 million people are infected and approximately 5% of these individuals will develop ATL, a highly aggressive cancer that is currently incurable. In the last years, genome editing tools have emerged as promising antiviral agents. In this proof-of-concept study, we use substrate-linked directed evolution (SLiDE) to engineer Cre-derived site-specific recombinases to excise the HTLV-1 proviral genome from infected cells. We identified a conserved loxP-like sequence (loxHTLV) present in the long terminal repeats of the majority of virus isolates. After 181 cycles of SLiDE, we isolated a designer-recombinase (designated RecHTLV), which efficiently recombines the loxHTLV sequence in bacteria and human cells with high specificity. Expression of RecHTLV in human Jurkat T cells resulted in antiviral activity when challenged with an HTLV-1 infection. Moreover, expression of RecHTLV in chronically infected SP cells led to the excision of HTLV-1 proviral DNA. Our data suggest that recombinase-mediated excision of the HTLV-1 provirus represents a promising approach to reduce proviral load in HTLV-1-infected individuals, potentially preventing the development of HTLV-1-associated diseases.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical , Adulto , Humanos , Vírus Linfotrópico T Tipo 1 Humano/genética , Paraparesia Espástica Tropical/tratamento farmacológico , Paraparesia Espástica Tropical/genética , Provírus/genética , Antivirais
3.
Nat Commun ; 13(1): 422, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35058465

RESUMO

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.


Assuntos
Inversão Cromossômica/genética , Fator VIII/genética , Recombinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular , Células Clonais , Evolução Molecular Direcionada , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Éxons/genética , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequências Repetidas Invertidas/genética , Recombinação Genética/genética , Especificidade por Substrato , Sequenciamento Completo do Genoma
4.
Nucleic Acids Res ; 50(2): 1174-1186, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34951450

RESUMO

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.


Assuntos
DNA Nucleotidiltransferases/metabolismo , Edição de Genes , Engenharia Genética/métodos , Recombinação Genética , Células HEK293 , Humanos
5.
Nucleic Acids Res ; 48(1): 472-485, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31745551

RESUMO

Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.


Assuntos
Sequência de Bases , Cromossomos Humanos Par 7/química , DNA Nucleotidiltransferases/genética , Edição de Genes/métodos , Genoma Humano , Deleção de Sequência , Cromossomos Humanos Par 7/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Loci Gênicos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Bio Protoc ; 8(1)2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29335676

RESUMO

Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O2) and carbon dioxide (CO2) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function.

7.
Sci Rep ; 7(1): 7294, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28779171

RESUMO

Development of complex nervous systems requires precisely controlled neurogenesis. The generation and specification of neurons occur through the transcriptional and post-transcriptional control of complex regulatory networks. In vertebrates and invertebrates, the proneural basic-helix-loop-helix (bHLH) family of transcription factors has multiple functions in neurogenesis. Here, we identified the LIN-32/Atonal bHLH transcription factor as a key regulator of URXL/R oxygen-sensing neuron development in Caenorhabditis elegans. When LIN-32/Atonal expression is lost, the expression of URX specification and terminal differentiation genes is abrogated. As such, lin-32 mutant animals are unable to respond to increases in environmental oxygen. The URX neurons are generated from a branch of the cell lineage that also produces the CEPDL/R and URADL/R neurons. We found development of these neurons is also defective, suggesting that LIN-32/Atonal regulates neuronal development of the entire lineage. Finally, our results show that aspects of URX neuronal fate are partially restored in lin-32 mutant animals when the apoptosis pathway is inhibited. This suggests that, as in other organisms, LIN-32/Atonal regulates neuronal apoptosis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Neurogênese , Neurônios/metabolismo , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Expressão Gênica , Genes Reporter , Mutação , Fatores de Transcrição/genética
8.
Sci Rep ; 7: 38734, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28139692

RESUMO

Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO2 sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO2 causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO2. Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO2-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed.


Assuntos
Caenorhabditis elegans/fisiologia , Dióxido de Carbono/metabolismo , Regulação da Expressão Gênica , Neuropeptídeos/biossíntese , Células Receptoras Sensoriais/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Receptores Acoplados a Guanilato Ciclase/metabolismo
9.
Genetics ; 199(1): 157-63, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25395666

RESUMO

Oxygen (O2) and carbon dioxide (CO2) provoke distinct olfactory behaviors via specialized sensory neurons across metazoa. In the nematode C. elegans, the BAG sensory neurons are specialized to sense changes in both O2 and CO2 levels in the environment. The precise functionality of these neurons is specified by the coexpression of a membrane-bound receptor-type guanylyl cyclase GCY-9 that is required for responses to CO2 upshifts and the soluble guanylyl cyclases GCY-31 and GCY-33 that mediate responses to downshifts in O2. Expression of these gas-sensing molecules in the BAG neurons is partially, although not completely, controlled by ETS-5, an ETS-domain-containing transcription factor, and EGL-13, a Sox transcription factor. We report here the identification of EGL-46, a zinc-finger transcription factor, which regulates BAG gas-sensing fate in partially parallel pathways to ETS-5 and EGL-13. Thereby, three conserved transcription factors collaborate to ensure neuron type-specific identity features of the BAG gas-sensing neurons.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Células Quimiorreceptoras/metabolismo , Neurogênese , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Dióxido de Carbono/farmacologia , Células Quimiorreceptoras/efeitos dos fármacos , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Oxigênio/farmacologia , Fatores de Transcrição/genética
10.
PLoS Genet ; 9(5): e1003511, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23671427

RESUMO

Animals harbor specialized neuronal systems that are used for sensing and coordinating responses to changes in oxygen (O2) and carbon dioxide (CO2). In Caenorhabditis elegans, the O2/CO2 sensory system comprises functionally and morphologically distinct sensory neurons that mediate rapid behavioral responses to exquisite changes in O2 or CO2 levels via different sensory receptors. How the diversification of the O2- and CO2-sensing neurons is established is poorly understood. We show here that the molecular identity of both the BAG (O2/CO2-sensing) and the URX (O2-sensing) neurons is controlled by the phylogenetically conserved SoxD transcription factor homolog EGL-13. egl-13 mutant animals fail to fully express the distinct terminal gene batteries of the BAG and URX neurons and, as such, are unable to mount behavioral responses to changes in O2 and CO2. We found that the expression of egl-13 is regulated in the BAG and URX neurons by two conserved transcription factors-ETS-5(Ets factor) in the BAG neurons and AHR-1(bHLH factor) in the URX neurons. In addition, we found that EGL-13 acts in partially parallel pathways with both ETS-5 and AHR-1 to direct BAG and URX neuronal fate respectively. Finally, we found that EGL-13 is sufficient to induce O2- and CO2-sensing cell fates in some cellular contexts. Thus, the same core regulatory factor, egl-13, is required and sufficient to specify the distinct fates of O2- and CO2-sensing neurons in C. elegans. These findings extend our understanding of mechanisms of neuronal diversification and the regulation of molecular factors that may be conserved in higher organisms.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Dióxido de Carbono/metabolismo , Dióxido de Carbono/fisiologia , Mutação , Oxigênio/metabolismo , Oxigênio/fisiologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Células Receptoras Sensoriais/citologia , Fatores de Transcrição/metabolismo
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