Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.007
Filtrar
1.
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
2.
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
3.
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
4.
PLoS One ; 15(7): e0236666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735574

RESUMO

Deciphering long-range chromatin interactions is critical for understanding temporal and tissue-specific gene expression regulated by cis- and trans-acting factors. By combining the chromosome conformation capture (3C) and biotinylated dCas9 system, we previously established a method CAPTURE-3C-seq to unbiasedly identify high-resolution and locus-specific long-range DNA interactions. Here we present the statistical model and a flexible pipeline, C3S, for analysing CAPTURE-3C-seq or similar experimental data from raw sequencing reads to significantly interacting chromatin loci. C3S provides all steps for data processing, quality control and result illustration. It can automatically define the bin size based on the binding peak of the dCas9-targeted regions. Furthermore, it supports the analysis of intra- and inter-chromosomal interactions for different mammalian cell types. We successfully applied C3S across multiple datasets in human K562 cells and mouse embryonic stem cells (mESC) for detecting known and new chromatin interactions at multiple scales. Integrative and topological analysis of the interacted loci at the human ß-globin gene cluster provides new insights into mechanisms in developmental gene regulation and network structure in local chromosomal architecture. Furthermore, computational results in mESCs reveal a role for chromatin interacting loops between enhancers and promoters in regulating alternative transcripts of the pluripotency gene OCT4.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Cromatina/química , Cromatina/metabolismo , Modelos Moleculares , Cromatina/genética , Ligação Proteica , Conformação Proteica
5.
PLoS Genet ; 16(8): e1008953, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776944

RESUMO

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.


Assuntos
Proteínas de Ligação a DNA/genética , Surdez/genética , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Benzotiazóis/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteínas de Ligação a DNA/deficiência , Surdez/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas Internas/patologia , Humanos , Camundongos , Mutação , RNA Guia/genética , Transdução de Sinais/efeitos dos fármacos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
6.
PLoS One ; 15(8): e0235942, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804931

RESUMO

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.


Assuntos
Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mutação INDEL , Solanum tuberosum/genética , Staphylococcus aureus/enzimologia , Sistemas CRISPR-Cas , Mutação da Fase de Leitura , Edição de Genes/métodos , Genoma de Planta , Plasmídeos/genética , Staphylococcus aureus/genética
7.
Yakugaku Zasshi ; 140(8): 993-1000, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741873

RESUMO

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Engenharia Genética , Luz , Optogenética , Ativação Transcricional , Animais , Diferenciação Celular , DNA/genética , Edição de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Recombinação Genética
8.
Nat Commun ; 11(1): 4043, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792475

RESUMO

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a ß-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Engenharia Celular/métodos , Biologia Sintética/métodos , Proteína 9 Associada à CRISPR/genética , Linhagem Celular , Edição de Genes , Humanos , Sistemas de Infusão de Insulina
9.
Rev. bioét. derecho ; (49): 77-91, jul. 2020.
Artigo em Espanhol | IBECS | ID: ibc-192095

RESUMO

El objetivo de este artículo es comprender cómo el CRISPR-Cas9 puede funcionar como una tecnología viable en la construcción del proyecto de parentalidad para promover el ejercicio de la libertad en el proceso de autonomía reproductiva. Sin embargo, a pesar de la posibilidad del uso en la línea germinal humana, se intentó investigar los límites y el alcance de esta percepción de libertad. Así, a través del uso de investigaciones documentales y bibliográficas, se buscaron datos sobre la viabilidad de la preservación del patrimonio genético como expresión de la diversidad en la humanidad. De esta forma, se percibió que es necesario superar pensamientos higienistas de limpiar "defectos", considerando que toda vida es digna de ser vivida


The scope of this paper was to understand how CRISPR-Cas9 can function as a viable technology in the construction of the parenting project in order to promote freedom in the process of procreative autonomy. However, regarding the possibility of its use to cause genetic manipulation in the human germline, we sought to ascertain the limits and scope of this perception of freedom. Thus, through the use of documentary and bibliographical method, data about the viability of the preservation of the genetic patrimony as an expression of the diversity in humanity was researched. Therefore, it was perceived as necessary to overcome the hygienist thoughts of cleaning up "defects", considering all life as worthy of being lived


L'objectiu del article era comprendre com CRISPR-CAS9 pot funcionar com una tecnologia viable en la construcció del projecte de parentalitat per promoure l'exercici de la llibertat en el procés d'autonomia reproductiva. No obstant això, tot I la possibilitat del seu ús en la línia germinal humana, es van intentar investigar els límits I l'abast d'aquesta percepció de llibertat. Així, a través de l'ús d'investigacions documentals I bibliogràfiques, es van buscar dades sobre la viabilitat de la preservació del patrimoni genètic com a expressió de la diversitat a la humanitat. D'aquesta manera, es va percebre que cal superar pensaments higienistes de netejar "defectes", considerant que tota vida és digna de ser viscuda


Assuntos
Humanos , Edição de Genes/ética , Proteína 9 Associada à CRISPR/genética , Bioética , Edição de Genes/legislação & jurisprudência , Direitos Humanos , Dissidências e Disputas/legislação & jurisprudência , Genoma Humano/genética , Genômica/ética , Pessoalidade
10.
PLoS Biol ; 18(7): e3000747, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644995

RESUMO

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Staphylococcus aureus/metabolismo , Biblioteca Gênica , Engenharia Genética , Loci Gênicos , Genoma Humano , Células HEK293 , Humanos , Nucleotídeos/genética , Fenótipo , Reprodutibilidade dos Testes , Ativação Transcricional/genética
11.
Mol Cell ; 79(2): 221-233.e5, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32603710

RESUMO

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Epigênese Genética , Edição de Genes , Técnicas de Silenciamento de Genes , Humanos , Nucleossomos/metabolismo , Xenopus laevis
12.
Nat Commun ; 11(1): 3576, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681021

RESUMO

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , DNA Bacteriano/metabolismo , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Cinética , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
13.
Nat Commun ; 11(1): 3784, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728052

RESUMO

The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.


Assuntos
Bacteriófagos/genética , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Edição de Genes/métodos , Interações Hospedeiro-Parasita/genética , Homologia de Sequência de Aminoácidos
14.
Nature ; 585(7824): 298-302, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32669707

RESUMO

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.


Assuntos
Nucléolo Celular/enzimologia , Nucléolo Celular/genética , DNA Ribossômico/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Ribossomos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/fisiologia , DNA Helicases/metabolismo , DNA Intergênico/genética , Humanos , Enzimas Multifuncionais/metabolismo , Biossíntese de Proteínas , Estruturas R-Loop , RNA Helicases/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Ribonuclease H/metabolismo , Ribossomos/química , Ribossomos/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia
15.
PLoS Genet ; 16(7): e1008924, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673314

RESUMO

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.


Assuntos
Quebras de DNA de Cadeia Dupla , Distrofia Miotônica/genética , Recombinação Genética , Repetições de Trinucleotídeos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos Fúngicos/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Conversão Gênica/genética , Genoma Humano/genética , Humanos , Distrofia Miotônica/patologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Expansão das Repetições de Trinucleotídeos/genética
16.
Nat Biotechnol ; 38(7): 856-860, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483363

RESUMO

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.


Assuntos
Adenina , Sistemas CRISPR-Cas/genética , Citosina , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Desoxirribonuclease I/genética , Humanos , RNA/genética
17.
Nat Biotechnol ; 38(7): 861-864, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483364

RESUMO

Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Citosina Desaminase/genética , Edição de Genes , Adenina/química , Citosina/química , Células HEK293 , Humanos , Mutação/genética , RNA/genética
18.
Nat Biotechnol ; 38(7): 865-869, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483365

RESUMO

We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes , Adenina/metabolismo , Adenosina Desaminase/genética , Citosina/metabolismo , Desoxirribonuclease I/genética , Genoma Humano/genética , Glicoproteínas/genética , Guanina/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas Nucleares/genética , RNA/genética
19.
Nat Struct Mol Biol ; 27(7): 678-682, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514175

RESUMO

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleoside adducts offer a confident and quantitative measure of direct RNA-protein interaction. Using RBS-ID, we profiled ~2,000 human RBSs and probed Streptococcus pyogenes Cas9 to discover residues important for genome editing.


Assuntos
Biologia Molecular/métodos , RNA/química , RNA/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Proteína 9 Associada à CRISPR/metabolismo , Cromatografia Líquida , Reagentes para Ligações Cruzadas/química , Células HeLa , Humanos , Ácido Fluorídrico/química , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/metabolismo , Espectrometria de Massas em Tandem
20.
Science ; 368(6496): 1265-1269, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527834

RESUMO

CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Clivagem do DNA/efeitos da radiação , Reparo do DNA/genética , Edição de Genes/métodos , Análise de Célula Única/métodos , Quebras de DNA de Cadeia Dupla , Células HEK293 , Histonas/metabolismo , Humanos , Luz , Proteína Homóloga a MRE11/genética , Imagem Óptica/métodos , Fosforilação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA