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1.
Proc Natl Acad Sci U S A ; 116(42): 20959-20968, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570623

RESUMO

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , Francisella/enzimologia , Edição de Genes , Animais , Proteína 9 Associada à CRISPR/genética , Catálise , DNA/química , DNA/metabolismo , Francisella/genética , Genoma , Humanos , Cinética , Especificidade por Substrato
2.
J Am Chem Soc ; 142(32): 13954-13965, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32658470

RESUMO

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.


Assuntos
Sistemas CRISPR-Cas/genética , RNA Nucleotidiltransferases/genética , RNA/genética , Química Click , Edição de Genes , Modelos Moleculares , RNA/química , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , RNA Guia de Cinetoplastídeos/genética
3.
Nat Commun ; 15(1): 5471, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38942756

RESUMO

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Francisella , Edição de Genes , Humanos , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Amaurose Congênita de Leber/genética , Streptococcus pyogenes/genética , Células HEK293 , Mutação , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Engenharia de Proteínas/métodos , Genoma Humano
4.
Cell Rep ; 42(10): 113177, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37751355

RESUMO

Embryonic stem cells (ESCs) can undergo lineage-specific differentiation, giving rise to different cell types that constitute an organism. Although roles of transcription factors and chromatin modifiers in these cells have been described, how the alternative splicing (AS) machinery regulates their expression has not been sufficiently explored. Here, we show that the long non-coding RNA (lncRNA)-associated protein TOBF1 modulates the AS of transcripts necessary for maintaining stem cell identity in mouse ESCs. Among the genes affected is serine/arginine splicing factor 1 (SRSF1), whose AS leads to global changes in splicing and expression of a large number of downstream genes involved in the maintenance of ESC pluripotency. By overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and AS upon its knockout, we describe local nuclear territories where these distinct events converge. Collectively, these contribute to the maintenance of mouse ESC identity.


Assuntos
Processamento Alternativo , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Processamento Alternativo/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias , Cromatina/metabolismo
5.
Biosens Bioelectron ; 183: 113207, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33866136

RESUMO

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.


Assuntos
Técnicas Biossensoriais , COVID-19 , Teste para COVID-19 , Humanos , RNA Viral , SARS-CoV-2 , Sensibilidade e Especificidade
6.
Curr Top Dev Biol ; 138: 73-112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32220299

RESUMO

Embryonic Stem cells are widely studied to elucidate the disease and developmental processes because of their capability to differentiate into cells of any lineage, Pervasive transcription is a distinct feature of all multicellular organisms and genomic elements such as enhancers and bidirectional or unidirectional promoters regulate these processes. Thousands of loci in each species produce a class of transcripts called noncoding RNAs (ncRNAs), that are well known for their influential regulatory roles in multiple biological processes including stem cell pluripotency and differentiation. The number of lncRNA species increases in more complex organisms highlighting the importance of RNA-based control in the evolution of multicellular organisms. Over the past decade, numerous studies have shed light on lncRNA biogenesis and functional significance in the cell and the organism. In this review, we focus primarily on lncRNAs affecting the stem cell state and developmental pathways.


Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/fisiologia , RNA Longo não Codificante/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Sequências Reguladoras de Ácido Nucleico
7.
Sci Rep ; 8(1): 7673, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29769662

RESUMO

Clustered miRNAs can affect functioning of downstream pathways due to possible coordinated function. We observed 78-88% of the miR-379/miR-656 cluster (C14MC) miRNAs were downregulated in three sub-types of diffuse gliomas, which was also corroborated with analysis from The Cancer Genome Atlas (TCGA) datasets. The miRNA expression levels decreased with increasing tumor grade, indicating this downregulation as an early event in gliomagenesis. Higher expression of the C14MC miRNAs significantly improved glioblastioma prognosis (Pearson's r = 0.62; p < 3.08e-22). ENCODE meta-data analysis, followed by reporter assays validated existence of two novel internal regulators within C14MC. CRISPR activation of the most efficient internal regulator specifically induced members of the downstream miRNA sub-cluster and apoptosis in glioblastoma cells. Luciferase assays validated novel targets for miR-134 and miR-485-5p, two miRNAs from C14MC with the most number of target genes relevant for glioma. Overexpression of miR-134 and miR-485-5p in human glioblastoma cells suppressed invasion and proliferation, respectively. Furthermore, apoptosis was induced by both miRs, individually and in combination. The results emphasize the tumor suppressive role of C14MC in diffuse gliomas, and identifies two specific miRNAs with potential therapeutic value and towards better disease management and therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , MicroRNAs/genética , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Invasividade Neoplásica , Fenótipo , Células Tumorais Cultivadas
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