RESUMO
Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.
Assuntos
COVID-19/diagnóstico , Avaliação de Programas e Projetos de Saúde , Saliva/virologia , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Normas Sociais , Inquéritos e Questionários , Universidades , Adulto JovemRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)-driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR-based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/diagnóstico , Neoplasias/genética , Animais , HumanosRESUMO
Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Ribonucleoproteínas/genética , Humanos , Ribonucleoproteínas/metabolismoRESUMO
Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , DNA/genética , Desulfovibrio vulgaris/genética , Endonucleases/genética , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/genética , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Desulfovibrio vulgaris/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Expressão Gênica , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Óperon , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Many bacteria and archaea possess an adaptive immune system consisting of repetitive genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Similar to RNAi pathways in eukaryotes, CRISPR-Cas systems require small RNAs for sequence-specific detection and degradation of complementary nucleic acids. Cas5 and Cas6 enzymes have evolved to specifically recognize and process CRISPR-derived transcripts into functional small RNAs used as guides by interference complexes. Our detailed understanding of these proteins has led to the development of several useful Cas6-based biotechnological methods. Here, we review the structures, functions, mechanisms, and applications of the enzymes responsible for CRISPR RNA (crRNA) processing, highlighting a fascinating family of endonucleases with exquisite RNA recognition and cleavage activities.
Assuntos
Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas/genética , Endorribonucleases/química , Relação Estrutura-Atividade , Bactérias/genética , Bactérias/imunologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Conformação Proteica , Dobramento de Proteína , RNA/química , RNA/genética , Interferência de RNA , Especificidade por SubstratoRESUMO
The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [PSI(+)]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.
Assuntos
Príons/fisiologia , Dobramento de Proteína , Controle de Qualidade , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Príons/química , Estresse FisiológicoRESUMO
In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Imageamento Tridimensional , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Subunidades Proteicas , Interferência de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismoRESUMO
Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.