Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Biotechnol ; 40(4): 539-545, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34711989

RESUMO

The ability to control translation of endogenous or exogenous RNAs in eukaryotic cells would facilitate a variety of biotechnological applications. Current strategies are limited by low fold changes in transgene output and the size of trigger RNAs (trRNAs). Here we introduce eukaryotic toehold switches (eToeholds) as modular riboregulators. eToeholds contain internal ribosome entry site sequences and form inhibitory loops in the absence of a specific trRNA. When the trRNA is present, eToeholds anneal to it, disrupting the inhibitory loops and allowing translation. Through optimization of RNA annealing, we achieved up to 16-fold induction of transgene expression in mammalian cells. We demonstrate that eToeholds can discriminate among viral infection status, presence or absence of gene expression and cell types based on the presence of exogenous or endogenous RNA transcripts.


Assuntos
Biossíntese de Proteínas , RNA , Animais , Mamíferos/genética , Biossíntese de Proteínas/genética , RNA Viral/genética
2.
Mol Cell ; 65(6): 1109-1121.e3, 2017 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-28306506

RESUMO

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Campylobacter jejuni/enzimologia , Endonucleases/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas Associadas a CRISPR/química , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Endonucleases/química , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Science ; 351(6271): 403-7, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26721684

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.


Assuntos
Sistemas CRISPR-Cas , Distrofina/genética , Éxons/genética , Terapia Genética/métodos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Deleção de Sequência
5.
Science ; 351(6271): 407-411, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26721686

RESUMO

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.


Assuntos
Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Células Satélites de Músculo Esquelético/metabolismo , Transdução Genética/métodos , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus , Modelos Animais de Doenças , Éxons , Mutação da Fase de Leitura , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , RNA Mensageiro/genética , Deleção de Sequência
6.
Cell ; 162(5): 1113-26, 2015 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-26317473

RESUMO

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.


Assuntos
Proteínas de Bactérias/química , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Engenharia Genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Alinhamento de Sequência , Streptococcus pyogenes/enzimologia
7.
Nature ; 520(7546): 186-91, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25830891

RESUMO

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Engenharia Genética/métodos , Genoma/genética , Staphylococcus aureus/enzimologia , Animais , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Colesterol/sangue , Colesterol/metabolismo , Marcação de Genes , Fígado/metabolismo , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/biossíntese , Pró-Proteína Convertases/sangue , Pró-Proteína Convertases/deficiência , Pró-Proteína Convertases/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/sangue , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Staphylococcus aureus/genética , Especificidade por Substrato
8.
Science ; 343(6175): 1246980, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24604203

RESUMO

Little is known about how human genetic variation affects the responses to environmental stimuli in the context of complex diseases. Experimental and computational approaches were applied to determine the effects of genetic variation on the induction of pathogen-responsive genes in human dendritic cells. We identified 121 common genetic variants associated in cis with variation in expression responses to Escherichia coli lipopolysaccharide, influenza, or interferon-ß (IFN-ß). We localized and validated causal variants to binding sites of pathogen-activated STAT (signal transducer and activator of transcription) and IRF (IFN-regulatory factor) transcription factors. We also identified a common variant in IRF7 that is associated in trans with type I IFN induction in response to influenza infection. Our results reveal common alleles that explain interindividual variation in pathogen sensing and provide functional annotation for genetic variants that alter susceptibility to inflammatory diseases.


Assuntos
Células Dendríticas/imunologia , Interação Gene-Ambiente , Interações Hospedeiro-Patógeno/genética , Fator Regulador 7 de Interferon/genética , Fatores de Transcrição STAT/genética , Adulto , Doenças Autoimunes/genética , Doenças Transmissíveis/genética , Células Dendríticas/efeitos dos fármacos , Escherichia coli , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Vírus da Influenza A , Interferon beta/farmacologia , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Transcriptoma , Adulto Jovem
9.
Methods Mol Biol ; 1114: 269-77, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24557909

RESUMO

The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819-823, 2013; Mali P et al., Science 339, 823-826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.


Assuntos
Genoma , Edição de RNA , Animais , Linhagem Celular , Clonagem Molecular , Reparo do DNA por Junção de Extremidades , Ordem dos Genes , Marcação de Genes/métodos , Vetores Genéticos , Humanos , Transfecção , Pequeno RNA não Traduzido
10.
Cell ; 156(5): 935-49, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24529477

RESUMO

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.


Assuntos
Proteínas Associadas a CRISPR/química , Cristalografia por Raios X , Endonucleases/química , RNA Bacteriano/química , Streptococcus pyogenes/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Endonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Bacteriano/metabolismo , Alinhamento de Sequência , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/metabolismo , Pequeno RNA não Traduzido
11.
Nat Protoc ; 8(11): 2281-2308, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24157548

RESUMO

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética/métodos , Genoma , Sequência de Bases , Técnicas de Cultura de Células , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Análise Mutacional de DNA , Reparo do DNA , Desoxirribonucleases/química , Desoxirribonucleases/genética , Técnicas de Genotipagem , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutagênese , Transfecção
12.
Cell ; 154(6): 1380-9, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-23992846

RESUMO

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.


Assuntos
Quebras de DNA de Cadeia Dupla , Marcação de Genes/métodos , Genoma , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Zigoto/metabolismo , Pequeno RNA não Traduzido
13.
Nat Biotechnol ; 31(9): 827-32, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23873081

RESUMO

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.


Assuntos
DNA/genética , Desoxirribonucleases/genética , Engenharia Genética/métodos , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases , Sequência de Bases , Dados de Sequência Molecular , Streptococcus pyogenes/genética , Pequeno RNA não Traduzido
14.
Science ; 339(6121): 819-23, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23287718

RESUMO

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.


Assuntos
Sistemas CRISPR-Cas , Clivagem do DNA , Engenharia Genética/métodos , Genoma/genética , Sequências Repetidas Invertidas/genética , Análise em Microsséries/métodos , Animais , Sequência de Bases , DNA/química , DNA/genética , Loci Gênicos , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , RNA/química , RNA/genética , Reparo de DNA por Recombinação , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética
15.
Environ Microbiol Rep ; 2(3): 373-80, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23766109

RESUMO

Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

16.
Appl Environ Microbiol ; 72(8): 5239-45, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16885271

RESUMO

Short nucleotide sequence repetitions in DNA can provide selective benefits and also can be a source of genetic instability arising from deletions guided by pairing between misaligned strands. These findings raise the question of how the frequency of deletion mutations is influenced by the length of sequence repetitions and by the distance between them. An experimental approach to this question was presented by the heat-sensitive phenotype conferred by pcaG1102, a 30-bp deletion in one of the structural genes for Acinetobacter baylyi protocatechuate 3,4-dioxygenase, which is required for growth with quinate. The original pcaG1102 deletion appears to have been guided by pairing between slipped DNA strands from nearby repeated sequences in wild-type pcaG. Placement of an in-phase termination codon between the repeated sequences in pcaG prevents growth with quinate and permits selection of sequence-guided deletions that excise the codon and permit quinate to be used as a growth substrate at room temperature. Natural transformation facilitated introduction of 68 different variants of the wild-type repeat structure within pcaG into the A. baylyi chromosome, and the frequency of deletion between the repetitions was determined with a novel method, precision plating. The deletion frequency increases with repeat length, decreases with the distance between repeats, and requires a minimum amount of similarity to occur at measurable rates. Deletions occurred in a recA-deficient background. Their frequency was unaffected by deficiencies in mutS and was increased by inactivation of recG.


Assuntos
Acinetobacter/genética , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Mutação , Deleção de Sequência , Acinetobacter/enzimologia , Acinetobacter/crescimento & desenvolvimento , Sequência de Bases , Meios de Cultura , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Plasmídeos/genética , Protocatecoate-3,4-Dioxigenase/genética , Protocatecoate-3,4-Dioxigenase/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Reprodutibilidade dos Testes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...