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1.
N Engl J Med ; 381(13): 1240-1247, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31509667

RESUMO

The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.).


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Infecções por HIV/terapia , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Receptores CCR5/genética , Adulto , Antirretrovirais/uso terapêutico , Contagem de Células Sanguíneas , Contagem de Linfócito CD4 , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Carga Viral
2.
BMC Plant Biol ; 19(1): 333, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370789

RESUMO

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.


Assuntos
Edição de Genes/métodos , Genes de Plantas/genética , Gliadina/genética , Glutens/genética , Triticum/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Eletroforese em Gel de Poliacrilamida , Glutens/imunologia , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas , Alinhamento de Sequência
3.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
4.
BMC Plant Biol ; 19(1): 311, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307375

RESUMO

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.


Assuntos
Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Genes de Plantas , RNA Guia , Soja/genética , Ácido alfa-Linoleico/genética , Agrobacterium/genética , Sistemas CRISPR-Cas , Marcadores Genéticos , Vetores Genéticos , Técnicas de Genotipagem , Padrões de Herança , Plantas Geneticamente Modificadas
5.
Microbiol Res ; 226: 1-9, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284938

RESUMO

Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.


Assuntos
Citrus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Interferência de RNA , Bactérias/genética , Bactérias/patogenicidade , Sistemas CRISPR-Cas , Citrus/microbiologia , Produtos Agrícolas , Fungos/genética , Fungos/patogenicidade , Edição de Genes/métodos , Engenharia Genética , Vírus/genética , Vírus/patogenicidade
6.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
8.
Life Sci ; 232: 116636, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295471

RESUMO

Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/tendências , Genoma , Humanos , Plantas/genética
9.
Nat Commun ; 10(1): 2883, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253768

RESUMO

A substantial number of mouse genes, about 25%, are embryonically lethal when knocked out. Using current genetic tools, such as the CRISPR-Cas9 system, it is difficult-or even impossible-to produce viable mice with heritable embryonically lethal mutations. Here, we establish a one-step method for microinjection of CRISPR reagents into one blastomere of two-cell embryos to generate viable chimeric founder mice with a heritable embryonically lethal mutation, of either Virma or Dpm1. By examining founder mice, we identify a phenotype and role of Virma in regulating kidney metabolism in adult mice. Additionally, we generate knockout mice with a heritable postnatally lethal mutation, of either Slc17a5 or Ctla-4, and study its function in vivo. This one-step method provides a convenient system that rapidly generates knockout mice possessing lethal phenotypes. This allows relatively easy in vivo study of the associated genes' functions.


Assuntos
Sistemas CRISPR-Cas , Embrião de Mamíferos/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Edição de Genes/métodos , Engenharia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mutação , RNA Guia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Nat Commun ; 10(1): 2866, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253785

RESUMO

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA , Edição de Genes/métodos , Reparo de DNA por Recombinação/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênese Insercional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
11.
Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050

RESUMO

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
12.
Nature ; 571(7764): 275-278, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181567

RESUMO

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , Transfecção
13.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
14.
Adv Mater ; 31(33): e1902575, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215123

RESUMO

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Nanopartículas/química , RNA Guia/química , RNA Mensageiro/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/métodos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Oxirredução , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Guia/administração & dosagem , RNA Mensageiro/administração & dosagem
15.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
17.
Nat Biotechnol ; 37(6): 626-631, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110355

RESUMO

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Adenina/química , Citosina/química , Humanos , Nucleotídeos/química , Nucleotídeos/genética , Plasmídeos/química , Plasmídeos/genética
18.
Gene ; 709: 8-16, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132514

RESUMO

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.


Assuntos
Ascomicetos/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Fúngico , Protoplastos/metabolismo , Transformação Genética , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/genética
19.
Nat Commun ; 10(1): 2127, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31073154

RESUMO

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.


Assuntos
Aptâmeros de Nucleotídeos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , DNA/genética , Ligantes
20.
Nat Commun ; 10(1): 2119, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31073172

RESUMO

Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Epigênese Genética , Células-Tronco Neurais/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica , Camundongos , Neuroglia/citologia , Neuroglia/fisiologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Fatores de Transcrição SOXB1/genética , Transcrição Genética/genética
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