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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360895

RESUMO

BACKGROUND: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients', matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. RESULTS: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. CONCLUSIONS: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Diabetes Mellitus Tipo 2/genética , Edição de Genes/métodos , Expressão Gênica , Resistência à Insulina/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Feminino , Redes Reguladoras de Genes , Hospitais Universitários , Humanos , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade
2.
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
3.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
4.
Nat Commun ; 12(1): 4922, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389729

RESUMO

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Perda de Heterozigosidade/genética , Deleção de Sequência , Células Cultivadas , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Metilação de DNA , Expressão Gênica , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
5.
Nat Genet ; 53(8): 1177-1186, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341563

RESUMO

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.


Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
6.
BMC Plant Biol ; 21(1): 363, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376154

RESUMO

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Pinus/genética , DNA de Plantas , Mutação INDEL , RNA Guia , Ribonucleoproteínas/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
10.
Nature ; 596(7871): 291-295, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321659

RESUMO

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Edição de Genes/métodos , Terapia Genética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/metabolismo , Éxons/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular Espinal/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/biossíntese , Progranulinas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
11.
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
12.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210100

RESUMO

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Edição de Genes/métodos , Proteínas de Plantas/metabolismo , Triticale/metabolismo , Sistemas CRISPR-Cas , Sistema Enzimático do Citocromo P-450/genética , Genes Reporter , Mutação INDEL , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Transfecção , Triticale/genética
14.
Commun Biol ; 4(1): 830, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215845

RESUMO

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteína 9 Associada à CRISPR/genética , DNA/genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos Testes
15.
Commun Biol ; 4(1): 834, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215850

RESUMO

The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.


Assuntos
Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Detecção Precoce de Câncer/métodos , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HEK293 , Humanos , Neoplasias/diagnóstico
16.
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
17.
Anticancer Res ; 41(8): 3731-3740, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281831

RESUMO

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.


Assuntos
Adenoviridae/genética , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Transfecção/métodos , Neoplasias da Bexiga Urinária/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/patologia
18.
Methods Mol Biol ; 2320: 235-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302662

RESUMO

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) are powerful tools for elucidating the pathology behind inherited cardiomyopathies. Genome editing technologies enable targeted genome replacement and the generation of isogenic hiPSCs, allowing investigators to precisely determine the roles of identified mutations. Here, we describe a protocol to obtain isogenic hiPSCs with the corrected allele via homology-directed repair (HDR) using CRISPR/Cas9 genome editing under feeder-free conditions. Seeding hiPSCs in a 24-well plate and conducting the initial evaluation using direct genomic sequencing after 1 week is cost- and time-effective. Following optimization of the protocol, sequence confirmation of the corrected HDR clone is completed within 21 days.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular , Células Clonais/citologia , Células Clonais/metabolismo , Eletroporação/métodos , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Puromicina/farmacologia , Reparo de DNA por Recombinação
19.
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
20.
Life Sci ; 282: 119790, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34245772

RESUMO

Alzheimer's disease is a chronic lifestyle ailment whose occurrence has come to light with the increasing life expectancy due to better healthcare. The patient burden for AD is set to double by the year 2060 and advancement in research is of utmost importance to combat this problem. AD is characterized by the pathological hallmarks of amyloid plaques and neurofibrillary tangles. The disease has been implicated to have a genetic predisposition. The current treatment strategies are at best ameliorative in nature and offer no substantive cure. Immunotherapeutic approaches employed have shown few therapeutic benefits but the accelerated approval of aducanumab by the US-FDA shows clinical benefit merit. In addition, newer therapeutic approaches are the need of the hour. This review aims to highlight the pathology of the disease, followed by an insight into newer approaches like stem cell therapy and gene editing, focusing on possible CRISPR mediated targets.


Assuntos
Doença de Alzheimer/terapia , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Animais , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Imunoterapia/métodos , Transplante de Células-Tronco/métodos
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