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1.
Acta Biochim Biophys Sin (Shanghai) ; 52(12): 1413-1419, 2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33201182

RESUMO

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.


Assuntos
Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Suínos/sangue , Febre Suína Africana/sangue , Febre Suína Africana/virologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteínas Associadas a CRISPR/biossíntese , Proteínas Associadas a CRISPR/isolamento & purificação , Sistemas CRISPR-Cas , China , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Viral/genética , Desoxirribonuclease I/genética , Endodesoxirribonucleases/biossíntese , Endodesoxirribonucleases/isolamento & purificação , Fluorescência , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/metabolismo , Sensibilidade e Especificidade
2.
Protein Expr Purif ; 169: 105588, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32006655

RESUMO

The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.


Assuntos
Proteínas Associadas a CRISPR , Porphyromonas gingivalis/genética , Proteínas Associadas a CRISPR/biossíntese , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/biossíntese , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas Recombinantes
3.
Nucleic Acids Res ; 43(12): 6038-48, 2015 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-26007654

RESUMO

The CRISPR-Cas prokaryotic 'adaptive immune systems' represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2-3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Carboidratos Epimerases/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Sítios de Ligação , Proteínas Associadas a CRISPR/biossíntese , Carboidratos Epimerases/genética , Proteína Receptora de AMP Cíclico/genética , Glucose/metabolismo , Mutação , Óperon , Pectobacterium/genética , Pectobacterium/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
4.
Biochem Soc Trans ; 41(6): 1468-74, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24256239

RESUMO

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and Cas (CRISPR-associated) proteins confer acquired resistance against mobile genetic elements in a wide range of bacteria and archaea. The phytopathogen Pectobacterium atrosepticum SCRI1043 encodes a single subtype I-F CRISPR system, which is composed of three CRISPR arrays and the cas operon encoding Cas1, Cas3 (a Cas2-Cas3 fusion), Csy1, Csy2, Csy3 and Cas6f (Csy4). The CRISPR arrays are transcribed into pre-crRNA (CRISPR RNA) and then processed by Cas6f to generate crRNAs. Furthermore, the formation of Cas protein complexes has been implicated in both the interference and acquisition stages of defence. In the present paper, we discuss the development of tightly controlled 'programmable' CRISPR arrays as tools to investigate CRISPR-Cas function and the effects of chromosomal targeting. Finally, we address how chromosomal targeting by CRISPR-Cas can cause large-scale genome deletions, which can ultimately influence bacterial evolution and pathogenicity.


Assuntos
Proteínas Associadas a CRISPR/biossíntese , Sistemas CRISPR-Cas/fisiologia , Ilhas Genômicas/genética , Pectobacterium/genética , Pectobacterium/metabolismo , RNA Bacteriano/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/imunologia , Pectobacterium/imunologia , RNA Bacteriano/genética
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