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1.
Life Sci ; 275: 119368, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33741417

RESUMO

Glioblastoma multiforme (GBM) is one of the worst brain tumors arising from glial cells, causing many deaths annually. Surgery, chemotherapy, radiotherapy and immunotherapy are used for GBM treatment. However, GBM is still an incurable disease, and new approaches are required for its successful treatment. Because mutations and amplifications occurring in several genes are responsible for the progression and aggressive behavior of GBM cells, genetic approaches are of great importance in its treatment. Small interfering RNA (siRNA) is a new emerging tool to silence the genes responsible for disease progression, particularly cancer. SiRNA can be used for GBM treatment by down-regulating genes such as VEGF, STAT3, ELTD1 or EGFR. Furthermore, the use of siRNA can promote the chemosensitivity of GBM cells. However, the efficiency of siRNA in GBM is limited via its degradation by enzymes, and its off-targeting effects. SiRNA-loaded carriers, especially nanovehicles that are ligand-functionalized by CXCR4 or angiopep-2, can be used for the protection and targeted delivery of siRNA. Nanostructures can provide a platform for co-delivery of siRNA plus anti-tumor drugs as another benefit. The prepared nanovehicles should be stable and biocompatible in order to be tested in human studies.


Assuntos
Neoplasias Encefálicas/terapia , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Glioblastoma/terapia , RNA Interferente Pequeno/uso terapêutico , Animais , Glioblastoma/metabolismo , Humanos
2.
Int J Mol Sci ; 22(2)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467049

RESUMO

Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.


Assuntos
Evolução Molecular Direcionada/métodos , Edição de Genes/métodos , Marcação de Genes/métodos , Mutagênese , Animais , Sistemas CRISPR-Cas , Plantas/genética
3.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443210

RESUMO

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.


Assuntos
Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Células Cultivadas , Códon de Terminação , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Feminino , Genoma , Células Germinativas , Masculino , Proteínas dos Microfilamentos/genética , Recombinação Genética
4.
Nat Commun ; 12(1): 472, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473139

RESUMO

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.


Assuntos
Alelos , Células Clonais , Marcação de Genes/métodos , Células-Tronco Hematopoéticas , Recombinação Genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Camundongos , Mutação , Reparo Gênico Alvo-Dirigido/métodos
5.
Methods Mol Biol ; 2238: 241-257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471336

RESUMO

Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.


Assuntos
Agrobacterium/genética , Marcação de Genes/métodos , Recombinação Homóloga , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Seleção Genética , Vetores Genéticos/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Transformação Genética
6.
Methods Mol Biol ; 2162: 243-260, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926387

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Marcação de Genes/métodos , Lentivirus/genética , Integração Viral , Vetores Genéticos/genética , Células HEK293 , Humanos
7.
Methods Mol Biol ; 2162: 261-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926388

RESUMO

Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Marcação de Genes/métodos , Células HEK293 , Humanos
8.
Sci Rep ; 10(1): 22271, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335280

RESUMO

CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp ß-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.


Assuntos
Jacarés e Crocodilos/genética , Peptídeos Catiônicos Antimicrobianos/genética , Sistemas CRISPR-Cas/genética , Peixes-Gato/genética , Animais , Peixes-Gato/crescimento & desenvolvimento , Edição de Genes , Técnicas de Introdução de Genes , Marcação de Genes/métodos , Genoma/genética , RNA Guia/genética , Reparo de DNA por Recombinação/genética
9.
PLoS Genet ; 16(12): e1008983, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315895

RESUMO

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.


Assuntos
Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Metilação de DNA , Edição de Genes/métodos , Marcação de Genes/métodos , Vírus de Plantas/genética , RNA Guia/genética , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Epigenoma , RNA de Transferência/genética , Ativação Transcricional
10.
Arch Virol ; 165(12): 2837-2846, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33025197

RESUMO

Pseudorabies virus (PRV) is a pig pathogen that causes substantial economic losses to the pig industry. Infection of host cells by PRV is mediated by the membrane proteins nectin1 and nectin2, which are presumed to be receptors for PRV infection. Here, we generated nectin1/2 knockout (KO) cells with the aim of establishing a PRV-resistant cell model. Nectin1 and 2 were ablated in PK15 cells by CRISPR/Cas9-mediated gene targeting. PRV infection in either nectin1 or nectin2 KO cells showed a significant reduction in viral growth compared with wild-type (WT) cells. We further simultaneously deleted nectin1 and nectin2 in PK15 cells and found that double KO cells showed no further increase in resistance to PRV compared with single gene-KO cells, despite being more resistant than WT. By investigating the cell entry steps of PRV infection, we found that nectin1 or/and nectin2 KO did not greatly affect virus attachment or internalization to cells but blocked cell-to-cell spread. Our results demonstrate that KO of either nectin1 or nectin2 confers PRV resistance to PK15 cells. This strategy could be applied to establish PRV-resistant pigs with nectin1/2 modifications to benefit the pig industry.


Assuntos
Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/fisiologia , Nectinas/genética , Pseudorraiva/virologia , Animais , Linhagem Celular , Marcação de Genes/métodos , Mutação , Suínos , Doenças dos Suínos/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
11.
PLoS One ; 15(9): e0238950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960926

RESUMO

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Toxinas Biológicas/genética , Alelos , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Homozigoto , Fenótipo , RNA Guia/genética , Toxinas Biológicas/metabolismo , Transgenes
12.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1659-1671, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924364

RESUMO

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.


Assuntos
Escherichia coli , Inativação Gênica , Marcação de Genes , Técnicas Genéticas , Temperatura , Escherichia coli/genética , Flagelos , Marcação de Genes/métodos , Mutação , Plasmídeos
13.
Gene ; 753: 144795, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32450202

RESUMO

The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.


Assuntos
Agricultura/métodos , Produtos Agrícolas/genética , Engenharia Genética/métodos , Biomarcadores , Biotecnologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Resistência à Doença/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Genoma de Planta/genética , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética
14.
Nat Med ; 26(4): 535-541, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32284612

RESUMO

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.


Assuntos
Anemia Falciforme/patologia , Edição de Genes , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , gama-Globinas/genética , Anemia Falciforme/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Estudos de Viabilidade , Feminino , Edição de Genes/métodos , Marcação de Genes/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Cultura Primária de Células , Proteínas Repressoras/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia , gama-Globinas/metabolismo
15.
Sci Rep ; 10(1): 6797, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321936

RESUMO

Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Here, we have investigated the localization and interactomes of endogenously tagged CK1δ and CK1ε during interphase and mitosis. CK1δ and CK1ε localize to centrosomes throughout the cell cycle, and in interphase cells to the nucleus, and in both a diffuse and punctate pattern in the cytoplasm. Also, for the first time, they were detected at the midbody during cell division. Mass spectrometry analysis identified a total of 181 proteins co-purifying with a Venus multifunctional (VM)-tagged CK1δ and/or CK1ε. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient endocytosis, was consistently one of the most abundant interacting partners. We demonstrate that GAPVD1 is a substrate of CK1δ/ε with up to 38 phosphorylated residues in vitro and in vivo. Wildtype and a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, were able to rescue defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1. Our results indicate that GAPVD1 is an important interacting partner and substrate of CK1δ/ε for endocytosis.


Assuntos
Sistemas CRISPR-Cas , Caseína Quinase Idelta/genética , Caseína Quinase Iépsilon/genética , Endocitose/genética , Marcação de Genes/métodos , Fosfoproteínas/genética , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Células HEK293 , Células HeLa , Humanos , Interfase/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Especificidade por Substrato
16.
PLoS One ; 15(3): e0230126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226034

RESUMO

The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.


Assuntos
Marcação de Genes , Recombinação Homóloga/genética , Cadeias Pesadas de Miosina/genética , Animais , Edição de Genes/métodos , Marcação de Genes/métodos , Técnicas de Genotipagem , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Transformação Genética
17.
Science ; 368(6488): 290-296, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32217751

RESUMO

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Marcação de Genes/métodos , Predisposição Genética para Doença , Células HEK293 , Humanos , Mutagênese , Domínios Proteicos , Especificidade por Substrato
18.
Biochem Pharmacol ; 175: 113911, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32173365

RESUMO

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , Inativação Gênica/fisiologia , Marcação de Genes/métodos , Sequências Repetidas Invertidas/fisiologia , Proteínas Tirosina Quinases/genética , Purinas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/administração & dosagem
19.
Sci Adv ; 6(6): eaay0187, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32076642

RESUMO

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Proteínas Recombinantes de Fusão , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Ativação Enzimática , Marcação de Genes/métodos , Genes Reporter , Loci Gênicos , Humanos , Cinética , Modelos Teóricos , Especificidade por Substrato
20.
Cancer Res ; 80(8): 1669-1680, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32060145

RESUMO

The role of the ataxia-telangiectasia-mutated (ATM) gene in human malignancies, especially in solid tumors, remains poorly understood. In the present study, we explored the involvement of ATM in transforming primary human cells into cancer stem cells. We show that ATM plays an unexpected role in facilitating oncogene-induced malignant transformation through transcriptional reprogramming. Exogenous expression of an oncogene cocktail induced a significant amount of DNA double-strand breaks in human fibroblasts that caused persistent activation of ATM, which in turn enabled global transcriptional reprogramming through chromatin relaxation, allowing oncogenic transcription factors to access chromatin. Consistently, deficiencies in ATM significantly attenuated oncogene-induced transformation of human cells. In addition, ATM inhibition significantly reduced tumorigenesis in a mouse model of mammary cancer. ATM and cellular DNA damage response therefore play a previously unknown role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells. SIGNIFICANCE: These findings uncover a novel pro-oncogenic role for ATM and show that contrary to established theory, ATM does not always function as a tumor suppressor; its function is however dependent on cell type.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Transformação Celular Neoplásica/genética , Reprogramação Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Células-Tronco Neoplásicas/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/patologia , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Fibroblastos/patologia , Técnicas de Inativação de Genes , Marcação de Genes/métodos , Genes p53 , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Ativação Transcricional , Transcriptoma/fisiologia , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Ensaio Tumoral de Célula-Tronco/métodos
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