RESUMO
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
Assuntos
Diferenciação Celular , Fatores de Transcrição , Humanos , Cromatina , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição/metabolismo , Atlas como AssuntoRESUMO
Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Loci Gênicos/genética , Genoma Humano/genética , Indóis/farmacologia , Melanoma/genética , RNA Longo não Codificante/genética , Sulfonamidas/farmacologia , Ativação Transcricional/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Sítio de Iniciação de Transcrição , VemurafenibRESUMO
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Silenciamento de Genes/métodos , Leptotrichia/enzimologia , RNA/genética , RNA/metabolismo , Biocatálise , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Escherichia coli/genética , Genes Reporter/genética , Células HEK293 , Humanos , Leptotrichia/genética , Células Vegetais/metabolismo , RNA/análise , Interferência de RNA , Estresse Fisiológico , Especificidade por SubstratoRESUMO
This corrects the article DOI: 10.1038/nature23451.
RESUMO
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
Assuntos
Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/análise , Vírus da Dengue/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , Ribonucleases/química , Zika virus/isolamento & purificação , Bactérias/patogenicidade , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/genética , Dengue/diagnóstico , Vírus da Dengue/genética , Humanos , Mutação , Neoplasias/genética , Clivagem do RNA , RNA Viral/genética , Zika virus/genética , Infecção por Zika virus/diagnósticoRESUMO
Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.