RESUMO
CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma Bacteriano , Pasteurella pneumotropica/genética , RNA Guia de Cinetoplastídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes/métodos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Pasteurella pneumotropica/enzimologia , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodobacteraceae/enzimologia , Rhodobacteraceae/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
SARS-CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS-CoV infection in cultured cells, which suggests that SARS development may be due to elastase-mediated, enhanced SARS-CoV infection in the lungs. To explore this possibility, we examined whether co-infection of mice with SARS-CoV and Pp, a low-pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co-infected with SARS-CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33~90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co-infection with SARS-CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.