Background

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Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant global public health concern due, in large part, to its widespread resistance to antibiotics (Wu et al., 2023; Alghamdi et al., 2023). First identified in 1961, MRSA is a strain of S. aureus that has acquired resistance to β-lactam antibiotics, including penicillins and cephalosporins (Gurung et al., 2020; Alghamdi et al., 2023; Ventola, 2015). This resistance makes infections much more difficult to treat. MRSA infections contribute to increased mortality, morbidity, longer hospital stays, and higher healthcare costs (Gurung et al., 2020).

Globally, MRSA was responsible for over 100,000 deaths in 2019 (Alghamdi et al., 2023). In the United States, MRSA is estimated to cause 11,285 deaths annually (Ventola, 2015). Historically, MRSA was predominantly associated with healthcare settings, often referred to as hospital-acquired MRSA (HA-MRSA), frequently causing postsurgical wound infections (Alghamdi et al., 2023; Gurung et al., 2020). However, community-acquired MRSA (CA-MRSA) infections have seen a rapid increase in the general population over the past decade, though this rise may be slowing (Ventola, 2015). Both the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) list MRSA as a serious and significant virulent bacterial threat (Alghamdi et al., 2023).

Mechanisms of Antibiotic Resistance in MRSA

Bacterial resistance to antibiotics develops when bacteria evolve mechanisms to evade drug effects (Habboush & Guzman, 2023):

• Altering antibiotic components to render them ineffective
• Exporting antibiotics out of the bacterial cell
• Modifying outer structures or receptors to prevent antibiotic attachment
• Genetic mutations that allow bacteria to survive antibiotic exposure

MRSA primarily resists β-lactam antibiotics through two main mechanisms (Alghamdi et al., 2023): production of β-lactamase, which deactivates antibiotics, and expression of the mecA gene, which encodes penicillin-binding protein 2a (PBP2a). PBP2a allows cell wall synthesis to continue even in the presence of drugs. Importantly, the SCCmec element carrying mecA can be transferred horizontally, spreading resistance.

Current Treatment Landscape and Challenges

• Traditional treatments include daptomycin, linezolid, and vancomycin (Alghamdi et al., 2023).
• Vancomycin remains highly effective, particularly in pediatrics, but must be monitored carefully to preserve its utility (Gurung et al., 2020).
• Second-line options like clindamycin, fusidic acid, and mupirocin exist, but resistance is a concern (Alghamdi et al., 2023).

Emerging Resistance

MRSA has developed resistance to daptomycin, linezolid, and vancomycin, often through mutations and single nucleotide polymorphisms (SNPs) within key genes and SCCmec machinery (Alghamdi et al., 2023). Daptomycin resistance is frequently associated with mutations in the mprF gene, leading to increased production of lysyl-PG, a positively charged phospholipid that repels cationic antibiotics. Linezolid resistance can arise from mutations in ribosomal proteins or 23S rRNA genes, or through the acquisition of resistance genes such as fexA or cfr (Alghamdi et al., 2023). These mechanisms can develop rapidly (Iguchi et al., 2016). A recent study of MRSA isolates from children (2016–2021) found that all isolates remained susceptible to vancomycin and linezolid (Wu et al., 2023). However, high resistance rates were observed for erythromycin (76.88%) and clindamycin (54.97%). The clindamycin resistance rate was particularly high (72.73%) in MRSA isolates from bone and joint infections in children, suggesting it may not be suitable for empirical treatment of osteomyelitis in such cases (Wu et al., 2023). Trends from 2016–2021 also showed increasing resistance to levofloxacin and TMP-SMX, while resistance to erythromycin, clindamycin, tetracycline, gentamicin, and rifampin decreased (Wu et al., 2023).

Our Solution

Our project is about engineering a CRISPR-Cas9 system to selectively destroy MRSA, using S. aureus as our model. We’ve designed guide RNAs to target and inhibit MraY and DnaG primase, two essential genes involved in cell wall formation and DNA replication. We aim to disrupt fundamental processes that are vital for bacterial survival.

Our Targets: MraY and DnaG primase

MraY enzyme: MraY is an essential bacterial membrane enzyme involved in peptidoglycan synthesis, making it a promising yet underexplored target for new antibacterial agents (Tomayo Berida et al., 2025; Nakaya et al., 2022). Topological analysis suggests MraY has 10 transmembrane segments and five cytoplasmic domains, with the latter potentially involved in substrate recognition and catalysis (Bouhss et al., 1999). Sphaerimicins, natural macrocyclic nucleoside inhibitors of MraY, have shown potential but are structurally complex (Nakaya et al., 2022). Recently, first-in-class 1,2,4-triazole-based non-nucleoside inhibitors of MraY were discovered (Tomayo Berida et al., 2025). Through structure-activity relationship (SAR) studies and structure-based drug design, one compound was optimized to achieve an IC50 of 25 µM against S. aureus MraY (MraYSA). These inhibitors also demonstrated broad-spectrum antibacterial activity against MRSA, E. faecium, VRE, and Mycobacterium tuberculosis (Tomayo Berida et al., 2025).

DnaG primase: Bacterial DnaG primase, an essential component of the bacterial replisome, is another promising but underdeveloped target (Ilic et al., 2018). Its inhibition selectively halts bacterial DNA replication, leading to bactericidal effects (Ilic et al., 2018). Importantly, bacterial primases differ structurally from human primase, offering opportunities for selective drug development. The C-terminal domain (CTD) of S. aureus DnaG primase interacts with the DnaB helicase, an interaction vital for DNA replication (Catazaro et al., 2017). Small molecules such as acycloguanosine, adenosine, and myricetin bind to a common pocket on the closed conformation of primase CTD, potentially preventing its interaction with DnaB helicase (Catazaro et al., 2017). Tegaserod has also been shown to inhibit RNA primer formation by S. aureus DnaG primase (Lacriola et al., 2017).

References

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• Bouhss, A., et al. (1999). Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis. Molecular Microbiology, 34(3), 576–585.
• Gurung, R., et al. (2020). Antibiotic resistance pattern of Staphylococcus aureus with reference to MRSA isolates from pediatric patients. Future Science OA, 6(4), FSO464.
• Habboush, Y., & Guzman, N. (2025). Antibiotic resistance. StatPearls Publishing.
• Ilic, S., et al. (2018). DnaG primase—A target for the development of novel antibacterial agents. Antibiotics, 7(3), 72 .
• Lacriola, C. J., et al. (2017). Inhibition of DNA replication in Staphylococcus aureus by tegaserod. Journal of Antibiotics, 70(8), 918–920.
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