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1.
bioRxiv ; 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37781618

RESUMO

Eukaryotic viruses assemble compartments required for genome replication, but no such organelles are known to be essential for prokaryotic viruses. Bacteriophages of the family Chimalliviridae sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of viral protein ChmA. Using the dRfxCas13d-based knockdown system CRISPRi-ART, we show that ChmA is essential for the E. coli phage Goslar life cycle. Without ChmA, infections are arrested at an early stage in which the injected phage genome is enclosed in a membrane-bound vesicle capable of gene expression but not DNA replication. Not only do we demonstrate that the phage nucleus is essential for genome replication, but we also show that the Chimalliviridae early phage infection (EPI) vesicle is a transcriptionally active, phage-generated organelle.

3.
Nat Chem Biol ; 17(9): 982-988, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34354262

RESUMO

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.


Assuntos
COVID-19/genética , Sistemas CRISPR-Cas/genética , RNA Viral/genética , SARS-CoV-2/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
medRxiv ; 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33791736

RESUMO

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

5.
Proc Natl Acad Sci U S A ; 117(46): 28784-28794, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33127759

RESUMO

Single-cell measurement techniques can now probe gene expression in heterogeneous cell populations from the human body across a range of environmental and physiological conditions. However, new mathematical and computational methods are required to represent and analyze gene-expression changes that occur in complex mixtures of single cells as they respond to signals, drugs, or disease states. Here, we introduce a mathematical modeling platform, PopAlign, that automatically identifies subpopulations of cells within a heterogeneous mixture and tracks gene-expression and cell-abundance changes across subpopulations by constructing and comparing probabilistic models. Probabilistic models provide a low-error, compressed representation of single-cell data that enables efficient large-scale computations. We apply PopAlign to analyze the impact of 40 different immunomodulatory compounds on a heterogeneous population of donor-derived human immune cells as well as patient-specific disease signatures in multiple myeloma. PopAlign scales to comparisons involving tens to hundreds of samples, enabling large-scale studies of natural and engineered cell populations as they respond to drugs, signals, or physiological change.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Expressão Gênica/genética , Humanos , Modelos Estatísticos , Modelos Teóricos , Análise de Sequência de RNA/métodos
6.
Nat Struct Mol Biol ; 24(10): 825-833, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28892041

RESUMO

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.


Assuntos
Sistemas CRISPR-Cas , Clostridiales/enzimologia , Endonucleases/química , Endonucleases/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Cristalografia por Raios X , Conformação Proteica
7.
Nat Methods ; 13(12): 1043-1049, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27776111

RESUMO

The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR-dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.


Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Engenharia Genética/métodos , Transcrição Gênica , Transcriptoma , Técnicas de Cultura de Células , Perfilação da Expressão Gênica , Células HEK293 , Humanos
8.
Elife ; 3: e03638, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-25030911

RESUMO

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.


Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Citidina Trifosfato/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expressão Gênica , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
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