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1.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
2.
Nat Commun ; 12(1): 2165, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846332

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs. Despite a compelling need towards predictive understanding of natural and engineered editing events, how the RNA sequence and structure determine the editing efficiency and specificity (i.e., cis-regulation) is poorly understood. We apply a CRISPR/Cas9-mediated saturation mutagenesis approach to generate libraries of mutations near three natural editing substrates at their endogenous genomic loci. We use machine learning to integrate diverse RNA sequence and structure features to model editing levels measured by deep sequencing. We confirm known features and identify new features important for RNA editing. Training and testing XGBoost algorithm within the same substrate yield models that explain 68 to 86 percent of substrate-specific variation in editing levels. However, the models do not generalize across substrates, suggesting complex and context-dependent regulation patterns. Our integrative approach can be applied to larger scale experiments towards deciphering the RNA editing code.


Assuntos
Adenosina Desaminase/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mutagênese/genética , Edição de RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Algoritmos , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Células HEK293 , Humanos , Aprendizado de Máquina , Modelos Genéticos , Mutação/genética , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Especificidade por Substrato
3.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805827

RESUMO

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.


Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , RNA Guia/química , Ribonucleoproteínas/química , Streptococcus pyogenes/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Edição de Genes/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Guia/genética , RNA Guia/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimologia
4.
Gene ; 784: 145595, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33775846

RESUMO

CRISPR/Cas9 is a powerful tool for genome editing. Several studies have been conducted to take the benefit of the versatile tool in the fission yeast Schizosaccharomyces pombe. However, the protocols for the CRISPR/Cas9 system proposed in previous studies are complicated in culture conditions compared to traditional genome editing methods. In this study, we introduced vectors for expression of sgRNA as well as Cas9, which employ natMX6 and bsdMX6 dominant selection markers. Using these materials, we examined nutritional conditions of cell cultures and found that nitrogen depletion introduced in previous methods does not affect the efficiency of genome editing. We found that bsdMX6-based plasmids enable us to skip any recovery steps before plating onto medium containing blasticidin S, unlike other antibiotic resistance selection markers. We thus propose easier transformation procedures with natMX6 and particularly bsdMX6 markers. We also simulate prescreening of mutants by genotyping with DNA endonucleases or proofreading PCR instead of relying on existing knowledge of mutant phenotypes. These materials and methods assist easy construction of S. pombe strains using CRISPR/Cas9, thereby accelerating seamless introduction of CRISPR/Cas9 to S. pombe researchers.


Assuntos
Proteína 9 Associada à CRISPR/genética , Meios de Cultura/química , RNA Guia/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Vetores Genéticos/genética , Nitrogênio/química , Mutação Puntual , Schizosaccharomyces/genética
5.
Nat Commun ; 12(1): 1692, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727557

RESUMO

CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Redes Reguladoras de Genes , Técnicas Genéticas , RNA Guia/genética
6.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670702

RESUMO

Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.


Assuntos
Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/metabolismo , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Diglicerídeos/metabolismo , Deleção de Genes , Células Intersticiais do Testículo/ultraestrutura , Ligantes , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Progesterona/metabolismo , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Transdução de Sinais , Esteroides/biossíntese
7.
J Vis Exp ; (168)2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33720123

RESUMO

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.


Assuntos
Proteína BRCA1/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Variação Genética , Sequência de Bases , Neoplasias da Mama/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Guia/genética
8.
J Vis Exp ; (168)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33720132

RESUMO

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliais/metabolismo , Edição de Genes , Mesoderma/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Imunofluorescência , Camundongos , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética
9.
Nat Commun ; 12(1): 1944, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782402

RESUMO

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.


Assuntos
Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Genoma de Planta , Oryza/genética , Agrobacterium tumefaciens , Alelos , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Produtos Agrícolas , Endodesoxirribonucleases/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Oryza/metabolismo , Plantas Geneticamente Modificadas , RNA Guia/genética , RNA Guia/metabolismo , Alinhamento de Sequência
10.
Mol Cell ; 81(7): 1553-1565.e8, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33662274

RESUMO

Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1 min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36 h of Cas9 activity or 2-4 h of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair.


Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , Dano ao DNA , Edição de Genes , RNA Guia/química , Proteína 9 Associada à CRISPR/metabolismo , RNA Guia/metabolismo
11.
Nat Genet ; 53(4): 529-538, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33753930

RESUMO

Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.


Assuntos
Mapeamento Cromossômico/métodos , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , Adulto , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Criança , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , RNA Guia/genética , RNA Guia/metabolismo
12.
Nat Commun ; 12(1): 1384, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654077

RESUMO

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Edição de Genes , Células HEK293 , Humanos , Motivos de Nucleotídeos/genética
13.
Nat Protoc ; 16(3): 1511-1547, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33547443

RESUMO

The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Humanos , Motivos de Nucleotídeos/genética , RNA Guia/genética , Projetos de Pesquisa
14.
Nat Commun ; 12(1): 1034, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589617

RESUMO

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , RNA Guia/genética , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Bases de Dados Genéticas , Doença de Fabry/genética , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia A/patologia , Humanos , Modelos Biológicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mutação , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
15.
ACS Appl Mater Interfaces ; 13(5): 6043-6052, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33525876

RESUMO

DNA methylation is a kind of a crucial epigenetic marker orchestrating gene expression, molecular function, and cellular phenotype. However, manipulating the methylation status of specific genes remains challenging. Here, a clustered regularly interspaced palindromic repeats-Cas9-based near-infrared upconversion-activated DNA methylation editing system (CNAMS) was designed for the optogenetic editing of DNA methylation. The fusion proteins of photosensitive CRY2PHR, the catalytic domain of DNMT3A or TET1, and the fusion proteins for CIBN and catalytically inactive Cas9 (dCas9) were engineered. The CNAMS could control DNA methylation editing in response to blue light, thus allowing methylation editing in a spatiotemporal manner. Furthermore, after combination with upconversion nanoparticles, the spectral sensitivity of DNA methylation editing was extended from the blue light to near-infrared (NIR) light, providing the possibility for remote DNA methylation editing. These results demonstrated a meaningful step forward toward realizing the specific editing of DNA methylation, suggesting the wide utility of our CNAMS for functional studies on epigenetic regulation and potential therapeutic strategies for related diseases.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Técnicas Genéticas , Raios Infravermelhos , Neoplasias da Glândula Tireoide/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/genética , Proteína 9 Associada à CRISPR/metabolismo , Sobrevivência Celular , Células Cultivadas , Metilação de DNA/genética , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Tamanho da Partícula , Propriedades de Superfície , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/terapia
16.
Nat Commun ; 12(1): 1244, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623024

RESUMO

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.


Assuntos
Diferenciação Celular , Leishmania mexicana/citologia , Leishmania mexicana/enzimologia , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sobrevivência Celular , Feminino , Flagelos/enzimologia , Deleção de Genes , Leishmaniose/parasitologia , Leishmaniose/patologia , Camundongos Endogâmicos BALB C , Modelos Biológicos , Mutação/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Psychodidae/parasitologia
17.
Nat Commun ; 12(1): 896, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563994

RESUMO

Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.


Assuntos
Sistemas CRISPR-Cas , Epigenômica/métodos , Histonas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Acetilação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/farmacologia , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Sulfonamidas/farmacologia , Ativação Transcricional
18.
Nucleic Acids Res ; 49(5): 2598-2608, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33591315

RESUMO

Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.


Assuntos
Reparo do DNA por Junção de Extremidades , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Proteína 9 Associada à CRISPR/metabolismo , Quebra Cromossômica , Desoxirribonucleotídeos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
19.
Methods Mol Biol ; 2260: 37-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405030

RESUMO

CRISPR/Cas9 technology enables rapid and efficient genome editing in a variety of experimental systems. Genome editing using CRISPR/Cas9 has become an increasingly popular genetic engineering tool due to (1) an extensive array of commercial ready-to-use CRIPSR/Cas9 systems, (2) improved efficiency of cell delivery, and (3) the possibility to do multigene editing. Here, we describe a method to introduce single gene disruption in lung bronchial epithelial cells. This approach can be used to study host factors important for pathogen interaction or to identify and study genetic markers determining susceptibility to fungal disease.


Assuntos
Brônquios/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Epiteliais/metabolismo , Fungos/patogenicidade , Edição de Genes , Inativação Gênica , Brônquios/microbiologia , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Humanos , RNA Guia/genética , RNA Guia/metabolismo
20.
ACS Appl Mater Interfaces ; 13(7): 7890-7896, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33513005

RESUMO

Nanodrug delivery systems are very promising for highly efficient anticancer drug delivery. However, the present nanosystems are commonly located in the cytoplasm and mediate uncontrolled release of drugs into cytosol, while a large number of anticancer drugs function more efficiently inside the nucleus. Here, we constructed a CRISPR-dCas9-guided and telomerase-responsive nanosystem for nuclear targeting and smart release of anticancer drugs. CRISPR-dCas9 technology has been employed to achieve conjugation of mesoporous silica nanoparticles (MSNs) with a high payload of the active anticancer drug, doxorubicin (DOX). A specifically designed wrapping DNA was used as a telomerase-responsive biogate to encapsulate DOX within MSNs. The wrapping DNA is extended in the presence of telomerase, which is highly activated in tumor cells, but not in normal cells. The extended DNA sequence forms a rigid hairpin-like structure and diffuses away from the MSN surface. CRISPR-dCas9 specifically targets telomere-repetitive sequences at the tips of chromosomes, facilitating the precise delivery of the nanosystem to the nucleus, and effective drug release triggered by telomerase that was enriched around telomeric repeats. This study provides a strategy and nanosystem for nuclear-targeted delivery and tumor-specific release of anticancer drugs that will maximize the efficiency of cancer cell destruction.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteína 9 Associada à CRISPR/química , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Telomerase/química , Antibióticos Antineoplásicos/química , Proteína 9 Associada à CRISPR/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Estrutura Molecular , Nanopartículas/metabolismo , Imagem Óptica , Tamanho da Partícula , Porosidade , Propriedades de Superfície , Telomerase/metabolismo
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