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1.
Methods Mol Biol ; 2577: 177-188, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173573

RESUMO

Epigenome editing is a powerful approach for the establishment of a chromatin environment with desired properties at a selected genomic locus, which is used to influence the transcription of target genes and to study properties and functions of gene regulatory elements. Targeted DNA methylation is one of the most often used types of epigenome editing, which typically aims for gene silencing by methylation of gene promoters. Here, we describe the design principles of EpiEditors for targeted DNA methylation and provide step-by-step guidelines for the realization of this approach. We focus on the dCas9 protein as the state-of-the-art DNA targeting module fused to 10×SunTag as the most frequently used system for editing enhancement. Further, we discuss different flavors of DNA methyltransferase modules used for this purpose including the most specific variants developed recently. Finally, we explain the principles of gRNA selection, outline the setup of the cell culture experiments, and briefly introduce the available options for the downstream DNA methylation data analysis.


Assuntos
Metilação de DNA , RNA Guia , Sistemas CRISPR-Cas/genética , Cromatina , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilases de Modificação do DNA/genética , Epigênese Genética , Edição de Genes , Metiltransferases/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Fatores de Transcrição/metabolismo
2.
Methods Mol Biol ; 2577: 189-195, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173574

RESUMO

Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Metilação de DNA , Epigênese Genética , Edição de Genes/métodos , Regulação da Expressão Gênica , Ativação Transcricional
3.
Methods Mol Biol ; 2577: 197-209, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173575

RESUMO

Regulation of epigenomic functions requires controlled site-specific alteration of epigenetic information. This can be achieved by using designed DNA-binding domains, associated with effector domains, that function as targeted transcription factors or epigenetic modifiers. These effectors have been employed to study the implications of epigenetic modifications, and sequence-specific targeting has been instrumental in understanding the effect of these modification on gene regulation. Ultimately, these tools could be used for therapeutic applications to revert the epigenetic aberrations that have been linked to various diseases. The ability to spatiotemporally control gene expression is especially important for precise regulation of the epigenomic state. In this chapter, we describe the protocol for achieving highly efficient small molecule-inducible transcriptional activation of endogenous mammalian genes, mediated by a dCas9-based system that recruits transcriptional activation domains binding to a chain of concatenated coiled-coil peptides.


Assuntos
DNA , Fatores de Transcrição , Animais , Sistemas CRISPR-Cas , Mamíferos/genética , Peptídeos/genética , Fatores de Transcrição/genética , Ativação Transcricional , Regulação para Cima
4.
Methods Mol Biol ; 2577: 211-226, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173576

RESUMO

Genome editing technologies can be diverted into artificial transcription activators. In particular, researchers have improved dCas9-based technologies by tandem-fusing or trans-accumulating effector domains. Previously, we developed a hierarchical effector accumulation system named "TREE," enabling robust activation of target genes even when strongly silenced. In this chapter, we describe our protocol to design, construct, and validate the TREE-mediated target gene activation in cultured human cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , Edição de Genes/métodos , Humanos , RNA Guia/genética , Fatores de Transcrição/genética , Ativação Transcricional
5.
Methods Mol Biol ; 2577: 229-240, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173577

RESUMO

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.


Assuntos
Endonucleases , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Mamíferos/metabolismo , Camundongos , RNA , Ativação Transcricional
6.
Methods Mol Biol ; 2577: 255-268, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173579

RESUMO

Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Desmetilação do DNA , Metilação de DNA , Epigênese Genética , Epigenoma , Edição de Genes/métodos , Camundongos
7.
Methods Mol Biol ; 2564: 185-201, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107342

RESUMO

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.


Assuntos
Sistemas CRISPR-Cas , DNA de Cadeia Simples , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Nucleotídeos , Ribonucleoproteínas/genética
8.
Talanta ; 251: 123795, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35952502

RESUMO

The development of a biosensing platform with high sensitivity, high specificity, and low cost for the detection of biomarkers, especially one that is programmable and universal, is critical for disease surveillance and diagnosis, yet it remains a difficulty. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free biosensor platform for sensitive and specific detection of disease-related protein, small molecule and nucleic acid. In this strategy, we designed an exonuclease III-mediated target cycle and released a universal trigger chain to stimulate the enzyme activity of CRISPR/Cas12a for additional signal amplification. The hydrolysis of ssDNA-templated silver nanoclusters (ssDNA-Ag NCs) as the reporter probe resulted in a significant decrease of fluorescence intensity. This biosensing platform can be flexibly used to the sensitive and specific determination of protein, small molecule, or microRNA in biological samples by simply transforming the target recognized sequences in the DNA hairpin. In this work, a new label-free sensing system used the fluorescent ssDNA-Ag NCs as the signal output does not need to be marked in advance and has no background signal. In addition, the method has the advantages of low cost, simple operation and high speed, and provides an innovative idea for the development of a powerful clinical diagnosis tool.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Biomarcadores , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas , DNA/genética , DNA de Cadeia Simples/genética , Limite de Detecção , Prata
9.
Talanta ; 251: 123784, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35988346

RESUMO

MicroRNAs are proposed novel biomarker for noninvasive diagnosis of cancer. miRNA-143 is reported to be associated with the development of prostate cancer. However, detection of miRNAs is still challenging due to their unique characteristics, such as small size and high sequence homology among family members. We here developed a gold nanoparticle (AuNP)-based visual assay that combines with CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (HRCA), which is called HRCA enhanced CRISPR/Cas12a-based assay (HECA) for sensitive detection of miRNA-143. The sequence-specific recognition character of CRISPR/Cas12a and HRCA signal amplification strategy enables the HECA outstanding specificity and sensitivity. In optimal condition, 1 fM miRNA-143 could be detected by naked eyes, and down to aM level with the aid of UV-Vis instrument. The diagnostic performance of the HECA for clinical samples was also evaluated based on the receiving operating characteristic algorithm (ROC), and our results suggest the miR-143 is a promising biomarker for noninvasive diagnosis of prostate cancer. This method is simple in operation and requires minimum instrument. We expect it to be widely applied in clinical diagnostics, especially in low-resource settings.


Assuntos
Nanopartículas Metálicas , MicroRNAs , Neoplasias da Próstata , Sistemas CRISPR-Cas , Ouro , Humanos , Masculino , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética
10.
Talanta ; 251: 123748, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35921742

RESUMO

In this work, an electrochemiluminescence (ECL) biosensor with dual signal enhancement was constructed and used for DNA adenine methylation methyltransferase (Dam MTase) detection. At present of Dam MTase, restriction endonuclease (DPnI) cleaves hairpin DNA (HP) and releases the HP stem end as a single strand that can activate CRISPR/Cas12a trans-cleavage activity. Assisted by trans-cleavage, the distance between the signal quenching factor ferrocene (Fc) and the ECL signal unit increased, and the repulsion between the signal unit and the Indium Tin Oxides (ITO) electrode decreased. The above results resulted in an enhanced ECL signal. ECL intensity has a good linear relationship with the logarithm of Dam MTase concentration in the range of 5-70 U/mL with a detection limit of 23.4 mU/mL. The proposed biosensor was successfully utilized to detect of Dam MTase in serum samples.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Adenina , Técnicas Biossensoriais/métodos , DNA , Metilação de DNA , Enzimas de Restrição do DNA , Índio , Metalocenos , Metiltransferases , Óxidos , Estanho
11.
Methods Mol Biol ; 2567: 39-62, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255694

RESUMO

Genetic editing of hematopoietic stem and progenitor cells can be employed to understand gene-function relationships underlying hematopoietic cell biology, leading to new therapeutic approaches to treat disease. The ability to collect, purify, and manipulate primary cells outside the body permits testing of many different gene editing approaches. RNA-guided nucleases, such as CRISPR, have revolutionized gene editing based simply on Watson-Crick base-pairing, employed to direct activity to specific genomic loci. Given the ease and affordability of synthetic, custom RNA guides, testing of precision edits or large random pools in high-throughput screening studies is now widely available. With the ever-growing number of CRISPR nucleases being discovered or engineered, researchers now have a plethora of options for directed genomic change, including single base edits, nicks or double-stranded DNA cuts with blunt or staggered ends, as well as the ability to target CRISPR to other cellular oligonucleotides such as RNA or mitochondrial DNA. Except for single base editing strategies, precise rewriting of larger segments of the genetic code requires delivery of an additional component, templated DNA oligonucleotide(s) encoding the desired changes flanked by homologous sequences that permit recombination at or near the site of CRISPR activity. Altogether, the ever-growing CRISPR gene editing toolkit is an invaluable resource. This chapter outlines available technologies and the strategies for applying CRISPR-based editing in hematopoietic stem and progenitor cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Oligonucleotídeos , Células-Tronco , RNA , DNA Mitocondrial
12.
Methods Mol Biol ; 2575: 25-38, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36301469

RESUMO

Nucleic acids are paving the way for advanced therapeutics. Unveiling the genome enabled a better understanding of unique genotype-phenotype profiling. Methods for engineering and analysis of nucleic acids, from polymerase chain reaction to Cre-Lox recombination, are contributing greatly to biomarkers' discovery, mapping of cellular signaling cascades, and smart design of therapeutics in precision medicine. Investigating the different subtypes of DNA and RNA via sequencing and profiling is empowering the scientific community with valuable information, to be used in advanced therapeutics, tracking epigenetics linked to disease. Recent results from the application of nucleic acids in novel therapeutics and precision medicine are very encouraging, demonstrating great potential to treat cancer, viral infections via inoculation (e.g., SAR-COV-2 mRNA vaccines), along with metabolic and genetic disorders. Limitations posed by challenges in delivery mode are being addressed to enable efficient guided-gene-programmed precision therapies. With the focus on genetic engineering and novel therapeutics, more precisely, in precision medicine, this chapter discusses the advance enabled by knowledge derived from these innovative branches of biotechnology.


Assuntos
COVID-19 , Ácidos Nucleicos , Humanos , Medicina de Precisão , Engenharia Genética/métodos , DNA , Sistemas CRISPR-Cas
13.
Methods Mol Biol ; 2575: 261-268, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36301479

RESUMO

Genome alteration results in several diseases for which therapeutics are limited. Gene editing provides a strong and potential alternative for the treatment of rare and genetic diseases. CRISPR-Cas9-based system is now being envisaged as a potential tool for the cure of genetic diseases. The RNA-guided nuclease, SaCas9 enzyme, along with its HF versions is widely employed for in vivo gene editing because of its small size and high efficiency. The current work summarizes the widely used and improved methods for in vivo manipulation of genes. The potential of CRISPR-Cas9-based systems can be harnessed to treat genetic diseases and holds great promise for therapeutic interventions in gene therapy. The in vivo gene editing poses a caveat in the form of delivery systems, the tissue in question, and several other factors. This work describes the methods which have been optimized to offer high efficiency, delivery, and gene editing in vivo.


Assuntos
Sistemas CRISPR-Cas , Staphylococcus aureus , Sistemas CRISPR-Cas/genética , Staphylococcus aureus/metabolismo , Edição de Genes/métodos , Terapia Genética , Endonucleases/genética
14.
Methods Mol Biol ; 2575: 241-260, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36301478

RESUMO

The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.


Assuntos
Sistemas CRISPR-Cas , Eucariotos , Humanos , Sistemas CRISPR-Cas/genética , Eucariotos/genética , RNA Guia/genética , RNA Guia/metabolismo , Edição de Genes/métodos , DNA/genética
15.
Methods Mol Biol ; 2562: 335-349, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272086

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool that enables editing of the axolotl genome. In this chapter, we will cover how to retrieve gene sequences, confirm annotation, design CRISPR targets, analyze indels, and screen for Crispant axolotls. This is a comprehensive guide on how to use CRISPR on your favorite gene and gain insights into its function.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Ambystoma mexicanum/genética , Genoma , Mutação , RNA Guia/genética
16.
Methods Mol Biol ; 2562: 351-368, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272087

RESUMO

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.


Assuntos
Ambystoma mexicanum , Sistemas CRISPR-Cas , Animais , Ambystoma mexicanum/genética , Sistemas CRISPR-Cas/genética , Alelos , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes
17.
Methods Mol Biol ; 2555: 205-212, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306089

RESUMO

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.


Assuntos
Bacillus subtilis , Bacteriófagos , Bacillus subtilis/genética , Sistemas CRISPR-Cas/genética , Metagenoma
18.
Methods Mol Biol ; 2589: 379-399, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255638

RESUMO

Oncolytic virotherapy represents an efficient immunotherapeutic approach for cancer treatment. Oncolytic viruses (OVs) promote antitumor responses through tumor-selective cell lysis and immune system activation. However, some tumor cell lines and primary tumors display resistance to therapy. Here we describe a protocol to identify novel host factors responsible for tumor resistance to oncolysis using an unbiased genome-wide CRISPR-Cas9 loss-of-function screening. Cas9-expressing tumor cells are transduced with a library of pooled single-guide RNA (sgRNA)-expressing lentiviruses that target all human genes to obtain a cell population where each cell is knocked out for a single gene. Upon OV infection, resistant cells survive, while sensitive cells die. The relative abundance of each genome-integrated sgRNA is measured by next-generation sequencing (NGS) in resistant and control cells. This protocol is amenable to uncover host factors involved in the resistance to different OVs in multiple tumor models.


Assuntos
Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Sistemas CRISPR-Cas/genética , RNA Guia/genética , Vírus Oncolíticos/genética , Neoplasias/genética , Neoplasias/terapia , Sequenciamento de Nucleotídeos em Larga Escala
19.
Methods Mol Biol ; 2589: 429-454, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255641

RESUMO

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.


Assuntos
Redes Reguladoras de Genes , Inibidores de Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Proteína 9 Associada à CRISPR , Expressão Gênica , Histona Desacetilases/genética , Sistemas CRISPR-Cas
20.
Talanta ; 252: 123837, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35987123

RESUMO

MicroRNA (miRNA) play a vital role in the pathological development of many diseases. It is considered to be the diagnosis and potential biomarkers of prognosis. Herein, we proposed Bis-enzyme cascade Platform by combining T7 RNA polymerase and CRISPR-Cas12a (BPTC) for a miRNA detection. In the proposed BPTC, the RNA to DNA conversion ability of phi29 amplification and trans-cleavage of CRISPR-Cas12a are combined. The target miRNA can be amplified after binding to the recognizer ssDNA, and then transcribed the CRISPR-derived RNA (crRNA) by T7 RNA polymerase. The produced crRNA can thereby be assembled by CRISPR-Cas12a and recognized with its target dsDNA, thus triggered its trans-cleavage towards surrounding fluorescent reporters, labeled with a fluorophore and a corresponding quenching group. Based on the bis-enzyme cascade system, the biosensor shows highly sensitivity and excellent specificity. Moreover, this study provided a novel all-in-one detect strategy for miRNA and may open a new idea for the design of CRISR-Cas-based miRNA biosensing platforms.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Sistemas CRISPR-Cas , MicroRNAs/genética , Clivagem do DNA , DNA de Cadeia Simples , DNA
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