Extracellular Vesicle Inspired Nano Vesicles for CRISPR-based Targeting of MRSA

About This Grant

Methicillin-resistant Staphylococcus aureus (MRSA) strains are antibiotic resistant bacteria that are the leading cause of healthcare associated infections in the United States and around the world. While research has focused on the development of new and more powerful antibiotics, the strains keep evolving resulting in ‘super bugs’ that are difficult to eradicate. Therefore, a key knowledge gap is the lack of an efficient approach that can target these superbugs ubiquitously with minimal effects on host cells. CRISPR-based gene editing can be a promising approach to address this knowledge gap. However, translation of CRISPR therapeutics faces a tremendous challenge as current technology focuses mainly on vector-based approaches to constitutively express CRISPR components (Cas9 enzyme and the targeting single guide RNA (sgRNA)) in recipient cells. This approach is fraught with logistical challenges as well as severe side effects. These are major hurdles to clinical translation. The translatability of CRISPR therapeutics can improve tremendously if the rhCas9 enzyme and the sgRNA can be delivered directly as a ribonucleoprotein (RNP) complex. However, this has not been achieved in bacteria. This application is an ambitious effort to address both the knowledge gap as well as the translational hurdle to CRISPR-based antimicrobials. Utilizing our foundational knowledge gained by studying extracellular vesicles (EVs) for the past decade, we hypothesize that: Artificial vesicles (AVs), mimicking the bioactivity of natural EVs can be used to deliver antibacterial CRISPR RNP complex to MRSA strains without ectopic activity on mammalian cells. To test this hypothesis, we propose two specific aims. Aim 1 will engineer artificial vesicles capable of bacterial entry with specific focus on MRSA strains found predominantly in the USA and Aim 2 will develop CRISPR RNPs targeting the conserved regions of 16s rRNA gene to produce vesicles that are capable of bactericidal activity upon entry into MRSA. This aim will also study the effects (absence of) the vesicles in representative mammalian cells as well as evaluate the efficacy of the strategy in a systemic model of mouse MRSA infection. Overall, successful completion of these aims will establish a system for delivering CRISPR-based therapeutics for combating antibiotic resistance super bugs.