Can SpRY recognize any PAM in human cells? / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.

Structure-based optimization and design of CRISPR protein xCas9 / 生物工程学报

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.

Evaluation of cytokines produced by β-hemolytic streptococcus in acute pharyngotonsillitis / Avaliação das citosinas produzidas por estreptococos β-hemolíticos em faringotonsilites agudas

INTRODUCTION: The most common pathogen in bacterial pharyngotonsillitis is group A β-hemolytic streptococcus, although groups B, C, F,and G have also been associated with pharyngotonsillitis.OBJECTIVE: To assess the levels of the cytokines TNF-α, IL-6,IL-4, and IL-10 in bacterial pharyngotonsillitis caused by group A and non-A (groups B, C, F and G) β-hemolytic streptococcus.METHODS: The study was conducted at a pediatric emergency care unit. The sample comprised children (5-9 years old) with acute bacterial pharyngotonsillitis diagnosed between December of 2011 and May of 2012. The research involved collection of blood samples from the patients, enzyme-linked immunosorbent assay detection of TNF-α, IL-6,IL-4, and IL-10, and collection of two oropharyngeal swabs for bacterial isolation. Additionally, the medical history of the study participants was also collected.RESULTS: In the studied group (mean age: 5.93 years), higher pharyngotonsillitis incidence was observed in the female gender (64.76%). Higher incidence of tonsillar exudates was observed with groups A and C. No statistically significant differences in cytokine levels were observed among groups. However, the group A and the control group showed a difference in the IL-6 level (p = 0.0016).CONCLUSIONS: The Groups A and C showed higher cytokine levels than the Groups B and control, suggesting similar immunological patterns.

INTRODUÇÃO: O patógeno mais comumente associado à faringotonsilite bacteriana é o estreptococo β-hemolítico do grupo A, a despeito dos grupos B, C, F e G terem também sido associados com a faringotonsilite.OBJETIVO: Determinar os níveis das citosinas TNF-α, IL-6, IL-4, e IL-10 na faringotonsilite bacteriana causada pelos estreptococos β-hemolíticos do grupo A e não-A (grupos B, C, F e G).MÉTODO: O estudo foi conduzido em uma emergência pediátrica. A amostra estudada compreendeu crianças (entre 5 e 9 anos) com faringotonsilite aguda bacteriana diagnosticada entre dezembro de 2011 e maio de 2012. A pesquisa envolveu a coleta de amostras sanguíneas dos pacientes, a detecção, através do ELISA, de TNF-α, IL-6, IL-4, and IL-10, além da coleta de dois swabs orofaríngeos para isolamento bacteriano. Adicionalmente foi coletada a história médica dos participantes do estudo.RESULTADOS: No grupo estudado (idade média: 5,93 anos), a maior incidência de faringotonsilite foi observada no gênero feminino (64,76%). Foram detectadas maiores incidências de exsudatos tonsilares nos grupos A e C. Não foram observadas diferenças estatisticamente significantes dos níveis de citosinas entre os grupos. Porém os grupos A e o controle mostraram diferença nos níveis de IL-6 (p = 0.0016).CONCLUSÕES: Os grupos A e C mostraram maiores níveis de citosinas que os grupos B e o controle, sugerindo mecanismos imunológicos similares.

Uso de antiinflamatorios nao-hormonais em crianças com doenças virais versus risco potencial para sindrome de Reye e doencas invasivas graves por estreptococos do grupo A / Nonsteroidal anti-inflammatory drugs use in children with viral diseases versus potential risks for Reyes's syndrome and invasive group A streptococal infections

Desde o inicio da decada de 1980, o uso de aspirina tem sido relacionado a sindrome de Reye e, na ultima decada, apos ter sido detectado um aumento na frequencia das doencas invasivas graves causadas por estreptococo beta-hemolitico do grupo A, tem sido publicados diversos artigos sobre a possivel relacao entre o uso de antiinflamatorios nao-hormonais em criancas com varicela e a ocorrencia de fasciite necrosante e sindrome do choque toxico. Neste artigo, a autora relata os...

Lectin mediated binding of Streptococcus pyogenes to human oropharyngeal mucosal epithelial cells.

The study was carried out to determine the role of lectins and sugars in the adhesion of S. pyogenes to human pharyngeal and buccal epithelial cells. In vitro adhesion assay has shown that Con A and Dolicos biflorus lectins inhibited the attachment of S. pyogenes to the oropharyngeal mucosal cells. Among different sugars used, N-acetyl-D-galactosamine and D-galactose have significantly blocked the binding of streptococci to PEC and BEC. These findings indicate that lectins and sugar molecules mediate the adhesion of S. pyogenes to the pharyngeal epithelial cells which may be important in the cellular pathogenesis of streptococcal infections which originate at the human oropharyngeal mucosa.