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1.
Nat Commun ; 13(1): 2833, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35595757

RESUMO

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Células HEK293 , Humanos , RNA/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
2.
Nat Hum Behav ; 4(9): 972-982, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848231

RESUMO

Despite the widespread implementation of public health measures, coronavirus disease 2019 (COVID-19) continues to spread in the United States. To facilitate an agile response to the pandemic, we developed How We Feel, a web and mobile application that collects longitudinal self-reported survey responses on health, behaviour and demographics. Here, we report results from over 500,000 users in the United States from 2 April 2020 to 12 May 2020. We show that self-reported surveys can be used to build predictive models to identify likely COVID-19-positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation; show a variety of exposure, occupational and demographic risk factors for COVID-19 beyond symptoms; reveal factors for which users have been SARS-CoV-2 PCR tested; and highlight the temporal dynamics of symptoms and self-isolation behaviour. These results highlight the utility of collecting a diverse set of symptomatic, demographic, exposure and behavioural self-reported data to fight the COVID-19 pandemic.


Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Adulto , Doenças Assintomáticas/epidemiologia , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/psicologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Aplicativos Móveis , Modelos Estatísticos , Pandemias/prevenção & controle , Pandemias/estatística & dados numéricos , Pneumonia Viral/diagnóstico , Pneumonia Viral/prevenção & controle , Pneumonia Viral/psicologia , SARS-CoV-2 , Estados Unidos/epidemiologia
3.
Nat Rev Microbiol ; 17(8): 513-525, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31165781

RESUMO

The principal function of CRISPR-Cas systems in archaea and bacteria is defence against mobile genetic elements (MGEs), including viruses, plasmids and transposons. However, the relationships between CRISPR-Cas and MGEs are far more complex. Several classes of MGE contributed to the origin and evolution of CRISPR-Cas, and, conversely, CRISPR-Cas systems and their components were recruited by various MGEs for functions that remain largely uncharacterized. In this Analysis article, we investigate and substantially expand the range of CRISPR-Cas components carried by MGEs. Three groups of Tn7-like transposable elements encode 'minimal' type I CRISPR-Cas derivatives capable of target recognition but not cleavage, and another group encodes an inactivated type V variant. These partially inactivated CRISPR-Cas variants might mediate guide RNA-dependent integration of the respective transposons. Numerous plasmids and some prophages encode type IV systems, with similar predicted properties, that appear to contribute to competition among plasmids and between plasmids and viruses. Many prokaryotic viruses also carry CRISPR mini-arrays, some of which recognize other viruses and are implicated in inter-virus conflicts, and solitary repeat units, which could inhibit host CRISPR-Cas systems.


Assuntos
Sistemas CRISPR-Cas , Evolução Molecular , Transferência Genética Horizontal , Sequências Repetitivas Dispersas , Recombinação Genética , Archaea/genética , Bactérias/genética , Bacteriófagos/genética , Elementos de DNA Transponíveis , Plasmídeos
4.
Science ; 363(6422): 88-91, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30523077

RESUMO

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.


Assuntos
Sistemas CRISPR-Cas , DNA/química , RNA Guia de Cinetoplastídeos/química , Ribonucleases/química , Bases de Dados de Proteínas , Desoxirribonucleases/química , Escherichia coli , Biblioteca Gênica , Conformação de Ácido Nucleico
5.
Mol Cell ; 70(2): 327-339.e5, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29551514

RESUMO

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , RNA Bacteriano/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Bases de Dados Genéticas , Escherichia coli/enzimologia , Escherichia coli/genética , Eubacterium/enzimologia , Eubacterium/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Secundária de Proteína , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ruminococcus/enzimologia , Ruminococcus/genética , Relação Estrutura-Atividade
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