Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.796
Filtrar
1.
Nat Commun ; 12(1): 5206, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471126

RESUMO

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.


Assuntos
Sistemas CRISPR-Cas , Escherichia coli/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano , Genoma Bacteriano , Plasmídeos , RNA Guia/genética
2.
Mol Cell ; 81(17): 3650-3658.e5, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34390675

RESUMO

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.


Assuntos
Epitopos/genética , Edição de Genes/métodos , Biblioteca de Peptídeos , RNA Guia/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Epitopos/imunologia , Humanos , Mutagênese/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Guia/imunologia
3.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
4.
Methods Mol Biol ; 2351: 321-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382198

RESUMO

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Clonagem Molecular , Ordem dos Genes , Marcação de Genes/métodos , Humanos , Células Secretoras de Insulina/metabolismo , RNA Guia/química , RNA Guia/genética
5.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445123

RESUMO

Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.


Assuntos
Aterosclerose/genética , Sistemas CRISPR-Cas/genética , Animais , DNA/genética , Epigênese Genética/genética , Edição de Genes/métodos , Expressão Gênica/genética , Genoma/genética , Humanos , RNA/genética , RNA Guia/genética
6.
Curr Protoc ; 1(8): e232, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34432381

RESUMO

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Mutação com Perda de Função , RNA Guia/genética
7.
Methods Mol Biol ; 2312: 225-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228293

RESUMO

The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.


Assuntos
Proteína 9 Associada à CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/efeitos da radiação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Cultura de Células , Reparo do DNA por Junção de Extremidades , Células HEK293 , Humanos , Mutação INDEL , Mutação Puntual , RNA Guia/genética , RNA Guia/metabolismo
8.
Methods Mol Biol ; 2312: 309-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228299

RESUMO

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.


Assuntos
Encéfalo/metabolismo , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Técnicas de Transferência de Genes , Animais , Encéfalo/embriologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Guia/genética , RNA Guia/metabolismo
9.
Methods Mol Biol ; 2287: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270031

RESUMO

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.


Assuntos
Endonucleases/metabolismo , Haploidia , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos , RNA Guia/genética , Sistemas CRISPR-Cas , Meios de Cultura , Endonucleases/genética , Edição de Genes , Engenharia Genética , Genoma de Planta , Homozigoto , Hordeum/embriologia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento
10.
Methods Mol Biol ; 2320: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302663

RESUMO

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Células Clonais , Meios de Cultura , Primers do DNA , Resistência a Medicamentos/genética , Eletroporação , Células-Tronco Embrionárias/citologia , Edição de Genes/métodos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Puromicina/farmacologia , RNA Guia/genética , Reparo de DNA por Recombinação/genética
11.
Nat Commun ; 12(1): 4476, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294706

RESUMO

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.


Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Edição de Genes , Bacteriófagos/enzimologia , Bacteriófagos/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Clivagem do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA Guia/genética , RNA Guia/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
12.
Nat Commun ; 12(1): 4219, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244505

RESUMO

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , Staphylococcus/enzimologia , Animais , Proteína 9 Associada à CRISPR/isolamento & purificação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Parvovirinae/genética , Engenharia de Proteínas , Ribonucleases , Staphylococcus/genética , Especificidade por Substrato , Síndromes de Usher/genética , Síndromes de Usher/terapia
13.
Nat Commun ; 12(1): 4203, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244519

RESUMO

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.


Assuntos
Processamento Alternativo , Cardiomiopatia Dilatada/genética , Miócitos Cardíacos/patologia , Proteínas de Ligação a RNA/genética , RNA-Seq/métodos , Sistemas CRISPR-Cas/genética , Cardiomiopatia Dilatada/patologia , Diferenciação Celular/genética , Linhagem Celular , Estudos de Viabilidade , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Proteínas Musculares/genética , Mutação , Isoformas de RNA/genética , RNA Guia/genética
14.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203807

RESUMO

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Reparo do DNA por Junção de Extremidades/genética , Sequência de Bases , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oligonucleotídeos/metabolismo , RNA Guia/genética , Deleção de Sequência/genética
15.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208669

RESUMO

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5'-truncated sgRNAs inhibited target recognition. These results suggest that a 5'-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5'-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5' end of truncated sgRNAs. Further, 5'-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5'-NG), which may enable whole-genome single-base editing.


Assuntos
Região 5'-Flanqueadora , Sistemas CRISPR-Cas , Edição de Genes , RNA Guia/genética , Proteína 9 Associada à CRISPR/metabolismo , Reparo de Erro de Pareamento de DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes/métodos , Genoma Microbiano , Genômica/métodos
16.
Methods Mol Biol ; 2320: 261-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302664

RESUMO

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Sequência de Bases , Causalidade , Células Cultivadas , Cromatografia Líquida/métodos , DNA/isolamento & purificação , Doxiciclina/farmacologia , Citometria de Fluxo , Estudos de Associação Genética , Vetores Genéticos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lentivirus/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Guia/genética , Transfecção
18.
Methods Mol Biol ; 2348: 175-187, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160807

RESUMO

Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Inativação Gênica , RNA Longo não Codificante/genética , Sítios de Ligação , Eletroporação , Técnicas de Silenciamento de Genes , Marcação de Genes , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Guia/genética
19.
Nat Commun ; 12(1): 3719, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140489

RESUMO

Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx4cv mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.


Assuntos
Sistemas CRISPR-Cas , Distrofina/genética , Edição de Genes/métodos , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Animais , Linhagem Celular , Dependovirus , Modelos Animais de Doenças , Distrofina/metabolismo , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia/genética , RNA-Seq
20.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073486

RESUMO

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Plantas Geneticamente Modificadas/genética , RNA Guia/genética , Tetraploidia , Tabaco/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...