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1.
GM Crops Food ; 12(1): 125-144, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-33079628

RESUMO

Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Edição de Genes , Doenças das Plantas/genética
2.
GM Crops Food ; 12(1): 57-70, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877304

RESUMO

Hybrid seeds of several important crops with supreme qualities including yield, biotic and abiotic stress tolerance have been cultivated for decades. Thus far, a major challenge with hybrid seeds is that they do not have the ability to produce plants with the same qualities over subsequent generations. Apomixis, an asexual mode of reproduction by avoiding meiosis, exists naturally in flowering plants, and ultimately leads to seed production. Apomixis has the potential to preserve hybrid vigor for multiple generations in economically important plant genotypes. The evolution and genetics of asexual seed production are unclear, and much more effort will be required to determine the genetic architecture of this phenomenon. To fix hybrid vigor, synthetic apomixis has been suggested. The development of MiMe (mitosis instead of meiosis) genotypes has been utilized for clonal gamete production. However, the identification and parental origin of genes responsible for synthetic apomixis are little known and need further clarification. Genome modifications utilizing genome editing technologies (GETs), such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (cas), a reverse genetics tool, have paved the way toward the utilization of emerging technologies in plant molecular biology. Over the last decade, several genes in important crops have been successfully edited. The vast availability of GETs has made functional genomics studies easy to conduct in crops important for food security. Disruption in the expression of genes specific to egg cell MATRILINEAL (MTL) through the CRISPR/Cas genome editing system promotes the induction of haploid seed, whereas triple knockout of the Baby Boom (BBM) genes BBM1, BBM2, and BBM3 cause embryo arrest and abortion, which can be fully rescued by male-transmitted BBM1. The establishment of synthetic apomixis by engineering the MiMe genotype by genome editing of BBM1 expression or disruption of MTL leads to clonal seed production and heritability for multiple generations. In the present review, we discuss current developments related to the use of CRISPR/Cas technology in plants and the possibility of promoting apomixis in crops to preserve hybrid vigor. In addition, genetics, evolution, epigenetic modifications, and strategies for MiMe genotype development are discussed in detail.


Assuntos
Apomixia , Sistemas CRISPR-Cas , Produtos Agrícolas/genética , Edição de Genes , Vigor Híbrido , Sementes
4.
Nat Commun ; 11(1): 4871, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978399

RESUMO

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.


Assuntos
Adenina/química , Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , RNA/metabolismo , Proteína 9 Associada à CRISPR , Citosina , DNA , Exoma , Genoma , Células HEK293 , Humanos , Edição de RNA
5.
Nature ; 585(7826): 603-608, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939090

RESUMO

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.


Assuntos
Éteres/metabolismo , Ferroptose , Peroxissomos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Éteres/química , Feminino , Edição de Genes , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Peroxidação de Lipídeos , Masculino , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Peroxissomos/genética
6.
Clin Dermatol ; 38(4): 462-466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32972604

RESUMO

The human genome project yielded a compendium of genetic material that has allowed rapid advancement both in the technique of whole exome sequencing and also in the ability to identify single gene defects. The next generation of genetics has investigated how these genes interact in the development of disease, identifying pathways of illness and end organ tissue abnormal development. From the knowledge of single genes and pathways of genodermatosis development arises the opportunity to produce genetic therapies. This contribution reviews some of the exciting, emerging genetic therapies in genodermatoses.


Assuntos
Dermatopatias Genéticas/terapia , Administração Tópica , Anticolesterolemiantes/administração & dosagem , Displasia Ectodérmica Anidrótica Tipo 1/diagnóstico , Displasia Ectodérmica Anidrótica Tipo 1/genética , Displasia Ectodérmica Anidrótica Tipo 1/terapia , Epidermólise Bolhosa/diagnóstico , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/terapia , Edição de Genes , Humanos , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Transdução de Sinais/genética , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/genética , Ustekinumab/uso terapêutico , Sequenciamento Completo do Exoma
7.
Mol Cell ; 79(5): 703-704, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888434

RESUMO

Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , Citosina , DNA de Cadeia Simples , Desaminação
8.
PLoS Negl Trop Dis ; 14(8): e0008627, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866158

RESUMO

The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.


Assuntos
Brugia Malayi/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transfecção/métodos , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA de Helmintos/genética , Feminino , Edição de Genes , Genoma , Larva/genética , Luciferases , Microfilárias/genética
9.
Nat Commun ; 11(1): 4884, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985507

RESUMO

Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Moreover, by targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses.


Assuntos
Tecnologia de Impulso Genético/métodos , Herpesviridae/genética , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/fisiologia , DNA Viral/genética , Edição de Genes , Herpesviridae/fisiologia , Humanos , Replicação Viral
10.
Nat Commun ; 11(1): 4593, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929070

RESUMO

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.


Assuntos
Alelos , Sistemas CRISPR-Cas/genética , Núcleo Celular/genética , Edição de Genes , Terapia de Substituição Mitocondrial , Animais , Sequência de Bases , Modelos Animais de Doenças , Nanismo/genética , Perda do Embrião/genética , Feminino , Marcação de Genes , Genes Dominantes , Impressão Genômica , Heterozigoto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Reprodutibilidade dos Testes , Fatores de Tempo
11.
Curr Protoc Plant Biol ; 5(3): e20117, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32865887

RESUMO

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.


Assuntos
Arabidopsis/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes
12.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
14.
Nat Commun ; 11(1): 4034, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788576

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.


Assuntos
Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Plaquetas/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Códon/genética , Feminino , Loci Gênicos , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Testes de Mutagenicidade , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
15.
Proc Natl Acad Sci U S A ; 117(34): 20803-20813, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32764148

RESUMO

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Betacoronavirus/fisiologia , Células Cultivadas , Infecções por Coronavirus , Ebolavirus/fisiologia , Edição de Genes , Humanos , Pandemias , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pneumonia Viral , Proteínas do Envelope Viral/genética
16.
Nat Commun ; 11(1): 4148, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811834

RESUMO

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.


Assuntos
Desoxirribonucleases/genética , Dependovirus/genética , Infecções Oculares/terapia , Edição de Genes/métodos , Herpes Simples/terapia , Herpesvirus Humano 1/genética , Latência Viral/genética , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecções Oculares/genética , Infecções Oculares/virologia , Feminino , Células HEK293 , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/virologia , RNA-Seq , Análise de Célula Única , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/virologia , Células Vero
17.
Nat Commun ; 11(1): 4022, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782248

RESUMO

One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Policetídeo Sintases/genética , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Estrutura Molecular , Família Multigênica/genética , Policetídeo Sintases/metabolismo , Sirolimo/química , Sirolimo/metabolismo , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo
18.
Science ; 369(6503): 566-571, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732424

RESUMO

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.


Assuntos
Adenina/química , Adenosina Desaminase/química , Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , Proteínas de Escherichia coli/química , Edição de Genes , Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , Microscopia Crioeletrônica , Desaminação , Proteínas de Escherichia coli/genética
19.
Nat Commun ; 11(1): 4178, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826895

RESUMO

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.


Assuntos
Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Gânglios Espinais/metabolismo , Edição de Genes/métodos , Proteínas de Ligação ao Ferro/genética , Organoides/metabolismo , Células Receptoras Sensoriais/metabolismo , Antioxidantes/farmacologia , Sistemas CRISPR-Cas , Diferenciação Celular , Cromatina/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Predisposição Genética para Doença/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Mitocôndrias/metabolismo , Organoides/efeitos dos fármacos , Organoides/patologia , Células Receptoras Sensoriais/patologia , Análise de Sequência de RNA , Transcriptoma
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