RESUMO
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
Assuntos
Sistemas CRISPR-Cas , Reprogramação Celular , Epigênese Genética , Epigenoma , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Diferenciação Celular , Ilhas de CpG , Metilação de DNA , Inativação Gênica , Código das Histonas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Bacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms.
Assuntos
Archaea/genética , Bactérias/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Engenharia Genética/métodos , Animais , Archaea/imunologia , Archaea/virologia , Bactérias/imunologia , Bactérias/virologia , DNA Viral/genética , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Plantas/genéticaRESUMO
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting â¼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.
Assuntos
Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endorribonucleases , Retroalimentação , Humanos , Modelos Moleculares , Proteínas Serina-Treonina Quinases , RNA Guia de Cinetoplastídeos/metabolismo , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.
Assuntos
Imunidade Adaptativa , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Bacteriano/imunologia , Endodesoxirribonucleases/imunologia , Endonucleases/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/imunologia , Memória Imunológica , Fatores Hospedeiros de Integração/imunologia , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores Hospedeiros de Integração/genética , Fatores Hospedeiros de Integração/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.
Assuntos
Imunidade Adaptativa , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/imunologia , Integrases/metabolismo , Sequência Rica em At/genética , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , DNA/química , DNA/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/virologia , Conformação de Ácido Nucleico , Especificidade por Substrato , Transposases/metabolismoRESUMO
Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
Assuntos
Imunidade Adaptativa , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Viral/genética , DNA Viral/imunologia , Integração Viral , Bacteriófago M13/genética , Bacteriófago M13/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/química , Domínio Catalítico , Cristalografia por Raios X , DNA Viral/química , DNA Viral/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Integrases/química , Integrases/metabolismo , Modelos Moleculares , Integração Viral/genética , Integração Viral/imunologiaRESUMO
Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Ativação Transcricional , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde/genética , Células HEK293 , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Proteínas Imediatamente Precoces/genética , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Transativadores/genética , Fator de Transcrição RelA/genética , TransfecçãoRESUMO
Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Proteômica , Pulmão , Células EpiteliaisRESUMO
SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Células Epiteliais/virologia , SARS-CoV-2/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , COVID-19/metabolismo , COVID-19/virologia , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Fatores de Transcrição/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.
RESUMO
Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
Assuntos
Fases de Leitura Aberta , Peptídeos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro , Sistemas CRISPR-Cas , Humanos , Óperon , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , TranscriptomaRESUMO
BACKGROUND: Transdermal lidocaine patches have few systemic toxicities and may be useful analgesics in cardiac surgery patients. However, few studies have evaluated their efficacy in the perioperative setting. Objective To compare the efficacy of topical lidocaine 5% patch plus standard care (opioid and nonopioid analgesics) with standard care alone for postthoracotomy or poststernotomy pain in adult patients in a cardiothoracic intensive care unit. METHODS: A single-center, retrospective cohort evaluation was conducted from January 2015 through December 2015 in the adult cardiothoracic intensive care unit at a tertiary academic medical center. Cardiac surgery patients with new sternotomies or thoracotomies were included. Patients in the lidocaine group received 1 to 3 topical lidocaine 5% patches near sternotomy and/or thoracotomy sites daily. Patches remained in place for 12 hours daily. Patients in the control group received standard care alone. RESULTS: The primary outcome was numeric pain rating for sternotomy/thoracotomy sites. Secondary outcomes were cardiothoracic intensive care unit and hospital lengths of stay and total doses of analgesics received. Forty-seven patients were included in the lidocaine group; 44 were included in the control group. Mean visual analogue scores for pain did not differ between groups (lidocaine, 2; control, 1.9; P = .58). Lengths of stay were similar for both groups (cardiothoracic intensive care unit: lidocaine, 3.06 days; control, 3.11 days; P = .86; hospital: lidocaine, 8.26 days; control, 7.61 days; P = .47). CONCLUSIONS: Adjunctive lidocaine 5% patches did not reduce acute pain in postthoracotomy and post-sternotomy patients in the cardiothoracic intensive care unit.
Assuntos
Analgésicos/uso terapêutico , Enfermagem de Cuidados Críticos/normas , Lidocaína/uso terapêutico , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/enfermagem , Esternotomia/efeitos adversos , Toracotomia/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Analgésicos/administração & dosagem , Estudos de Coortes , Método Duplo-Cego , Feminino , Humanos , Lidocaína/administração & dosagem , Masculino , Pessoa de Meia-Idade , Dor Pós-Operatória/etiologia , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Toracotomia/enfermagem , Adesivo TransdérmicoRESUMO
The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.
Assuntos
Sistemas CRISPR-Cas/fisiologia , Animais , Proteínas Associadas a CRISPR , Regulação da Expressão Gênica/fisiologia , Engenharia Genética , Genoma/genética , Ligação ProteicaRESUMO
The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.
Assuntos
Proteínas Associadas a CRISPR/fisiologia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Endodesoxirribonucleases/fisiologia , Endonucleases/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/imunologia , Imunidade Adaptativa , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Cristalografia por Raios X , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here we show that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9-Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate Lys27 of histone H3 in vitro and in vivo. Laser microirradiation data show that PHF1 is transiently recruited to DNA double-strand breaks, and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at double-strand break sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as a cofactor in early DNA-damage response.