Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

SUMMARYGroup A Streptococcus (GAS), also known as Streptococcus pyogenes, is a clinically well-adapted human pathogen that harbors rich virulence determinants contributing to a broad spectrum of diseases. GAS is capable of invading epithelial, endothelial, and professional phagocytic cells while evading host innate immune responses, including phagocytosis, selective autophagy, light chain 3-associated phagocytosis, and inflammation. However, without a more complete understanding of the different ways invasive GAS infections develop, it is difficult to appreciate how GAS survives and multiplies in host cells that have interactive immune networks. This review article attempts to provide an overview of the behaviors and mechanisms that allow pathogenic GAS to invade cells, along with the strategies that host cells practice to constrain GAS infection. We highlight the counteractions taken by GAS to apply virulence factors such as streptolysin O, nicotinamide-adenine dinucleotidase, and streptococcal pyrogenic exotoxin B as a hindrance to host innate immune responses.

OBJECTIVE: This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS).
METHODS: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out.
RESULTS: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria.
CONCLUSIONS: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

BACKGROUND: S. pyogenes, is a primary pathogen that leads to pharyngitis and can also trigger severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), often resulting in high mortality rates. Therefore, prompt identification and appropriate treatment of S. pyogenes infections are crucial in preventing the worsening of symptoms and alleviating the disease\'s impact.
RESULTS: In this study, a newly developed technique called multiple cross displacement amplification (MCDA) was employed to detect S. pyogenes,specifically targeting the speB gene, at a temperature of 63°C within 30 min. Then, an easily portable and user-friendly nanoparticles-based lateral flow biosensor (LFB) assay was introduced for the rapid analysis of MCDA products in just 2 min. The results indicated that the LFB offers greater objectivity compared to Malachite Green and is simpler than electrophoresis. The MCDA-LFB assay boasts a low detection limit of 200 fg and exhibits no cross-reaction with non-S. pyogenes strains. Among 230 clinical swab throat samples, the MCDA-LFB method identified 27 specimens as positive, demonstrating higher sensitivity compared to 23 samples detected positive by qPCR assay and 18 samples by culture. The only equipment needed for this assay is a portable dry block heater. Moreover, each MCDA-LFB test is cost-effective, priced at approximately $US 5.5.
CONCLUSIONS: The MCDA-LFB assay emerges as a straightforward, specific, sensitive, portable, and user-friendly method for the rapid diagnosis of S. pyogenes in clinical samples.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

-lactams, including penicillin, have been used for over 80 years in the treatment of group A Streptococcus (GAS) infections. Although -lactam-resistant GAS strains have not been identified in vitro tests, clinical treatment failures have been reported since the 1950s. The mechanism underlying the clinical failure of -lactam treatment in GAS infections remains unclear. Previous research has suggested that -lactam resistance in GAS in vivo is associated with reduced drug susceptibility of strains, bacterial inoculation effects, biofilm formation, the effect of coexisting bacteria, bacterial persistence, and bacterial internalization into host cells. This article reviews the main reports on -lactam treatment failure in GAS infections and analyzes the possible mechanisms of -lactam resistance in vivo. The findings aim to contribute to future research and clinical approaches in the field.
包括青霉素在内的-内酰胺类药物用于治疗A族链球菌（group A Streptococcus, GAS）感染已有80余年，虽然体外试验尚未发现对其耐药的GAS菌株，但自20世纪50年代以来，时有-内酰胺类药物临床治疗GAS感染失败的报道。GAS体内耐受-内酰胺类药物的机制尚未明确。已有研究提示，GAS体内耐受-内酰胺类药物与菌株对药物的敏感性降低、细菌的接种效应、生物膜的形成、共存菌的作用、细菌持留性及细菌可内化入胞内等因素有关。该文回顾总结了-内酰胺类药物临床治疗GAS感染失败的主要报道，以及与GAS体内耐受-内酰胺类药物机制相关的重要研究，为后续研究及临床用药提供参考。.

Streptococcus pyogenes (GAS) is one of the most virulent and infectious bacteria, severely threatening health and lives of people worldwide. Honey has been proven to have effective capability against GAS, but the underlying metabolites and mechanisms are still unclear. In this study, the Castanopsis honey (CH) showed significant antibacterial ability compared to other seven kinds of honey and artificial honey. Furthermore, the antibacterial metabolites and their targets in CH were screened by combined method of metabolomics, network pharmacology, and molecular docking. The results suggested that the activities of two antioxidant enzymes, glutathione peroxidase and tyrosyl tRNA synthetase identified as the primary targets, were significantly inhibited by CH, which significantly increased the level of oxidative stress in GAS. The results revealed a possibly novel mechanism regulating the oxidative stress and inhibits the growth in bacteria, providing strong experimental evidence to support the further development of CH as a novel antibacterial agent.

Streptococcal infection is a common cause of acute glomerulonephritis. Cardiac damage associated with streptococcal infection commonly occurs in acute rheumatic fever. However, cases of acute non-rheumatic streptococcal myocarditis have been reported in recent years. We report a novel case of concurrent acute glomerulonephritis and non-rheumatic myocarditis following streptococcal infection. A good prognosis was achieved with antibiotic and immunosuppressive therapy, indicating that Streptococcus causes cardiorenal syndrome type 5 via an immune-mediated response. A better understanding of post-streptococcal cardiorenal syndrome is warranted to enable the early diagnosis and treatment of affected patients.

The erythromycin polyketide compound TMC-154 is a secondary metabolite that is isolated from the rhizospheric fungus Clonostachys rogersoniana associated with Panax notoginseng, which possesses antibacterial activity. However, its antibacterial mechanism has not been investigated thus far. In this study, proteomics coupled with bioinformatics approaches was used to explore the antibacterial mechanism of TMC-154. KEGG pathway enrichment analysis indicated that eight signaling pathways were associated with TMC-154, including oxidative phosphorylation, cationic antimicrobial peptide (CAMP) resistance, benzoate degradation, heme acquisition systems, glycine/serine and threonine metabolism, beta-lactam resistance, ascorbate and aldarate metabolism, and phosphotransferase system (PTS). Cell biology experiments confirmed that TMC-154 could induce reactive oxygen species (ROS) generation in Streptococcus pyogenes; moreover, TMC-154-induced antibacterial effects could be blocked by the inhibition of ROS generation with the antioxidant N-acetyl L-cysteine. In addition, TMC-154 combined with ciprofloxacin or chloramphenicol had synergistic antibacterial effects. These findings indicate the potential of TMC-154 as a promising drug to treat S. pyogenes infections. SIGNIFICANCE: Streptococcus pyogenes is a nearly ubiquitous human pathogen that causes a variety of diseases ranging from mild pharyngitis and skin infection to fatal sepsis and toxic heat shock syndrome. With the increasing incidence of known antibiotic resistance, there is an urgent need to find novel drugs with good antibacterial activity against S. pyogenes. In this study, we found that TMC-154, a secondary metabolite from the fungus Clonostachys rogersoniana, inhibited the growth of various bacteria, including Staphylococcus aureus, S. pyogenes, Streptococcus mutans, Pseudomonas aeruginosa and Vibrio parahemolyticus. Proteomic analysis combined with cell biology experiments revealed that TMC-154 stimulated ROS generation to exert antibacterial effects against S. pyogenes. This study provides potential options for the treatment of S. pyogenes infections in the future.

Streptococcal toxic shock syndrome (STSS) is a severe consequence of infections from Streptococcus pyogenes. The early identification and timely intervention with appropriate anti-infective agents are pivotal for managing pediatric STSS. This study evaluates the effectiveness of various treatment regimens for STSS in children.
Clinical data of children with STSS resulting from β-hemolytic streptococcal infections in two hospitals were retrospectively analyzed from January 2009 to April 2023. Additionally, literature from the China National Knowledge Infrastructure on pediatric STSS was examined. Antimicrobial treatments were categorized into four groups based on their compositions, with an additional categorization for adjunct therapeutic drugs.
Of 32 confirmed STSS cases, all displayed sensitivity to ampicillin, β-lactam antibiotics, and vancomycin, but resistance to clindamycin, erythromycin, and tetracycline. From the literature, 23 studies with 50 cases were extracted, leading to a total of 82 patients for evaluation. The efficacy rates varied significantly among the four treatment groups. Notably, the standard penicillin-containing group exhibited the highest efficacy (86.4%), while the group with macrolides/unused antibiotics registered a 0% efficacy rate. The other two groups demonstrated efficacy rates of 32.1% and 42.3%.
For pediatric STSS, Streptococcus pyogenes shows notable sensitivity to ampicillin. Implementing timely β-lactam antibiotics, specifically penicillin, in conjunction with clindamycin and intravenous immunoglobulins enhances the treatment success rate.