Suppression of Antimicrobial Resistance in MRSA Using CRISPR-dCas9

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are genetic elements that function with CRISPR-Associated (Cas) proteins as an adaptive immune system to foreign genetic material in prokaryotic organisms. The CRISPR-dCas9 system is modified to suppress gene transcription. Methicillin-Resistant Staphylococcus aureus (MRSA) is a dangerous human pathogen that is resistant to beta-lactam antibiotics. This is due to the mecA methicillin resistance gene coding for penicillin binding protein 2A (PBP 2A), which inhibits the activity of beta-lactam antibiotics. Two CRISPR-dCas9 systems were designed to target the promoter region of mecA in MRSA to suppress transcription of the gene. A cefoxitin disk diffusion test showed that the target on the coding strand significantly reduced antibiotic resistance in MRSA, whereas the target on the noncoding strand did not. An oxacillin microbroth serial dilution was used to confirm disk diffusion results. The CRISPR system targeting the coding strand was the only one to reduce antibiotic resistance and thus was chosen for continued testing. mecA gene expression levels were analyzed using Reverse Transcriptase Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). Results showed that mecA gene expression in the CRISPR-treated sample was reduced to 0.230 fold of the value in the control, representing a 77% decrease in gene transcription. The 77% decrease in gene expression was not enough to make MRSA clinically susceptible to betalactam antibiotics. ABBREVIATIONS: BLAST - basic local alignment search tool, bp - basepairs, Cas - CRISPR-associated, cDNA - complementary DNA, CLSI - Clinical Laboratory Standards Institute, Cq - quantification cycle, CRISPRs - clustered regularly spaced short palindromic repeats, E.coli - Escherichia coli, M-MLV RT - Moloney Murine Leukemia Virus Reverse Transcriptase, MIC - minimum inhibitory concentration, MRSA - methicillin-resistant Staphylococcus aureus, NCBI - National Center for Biotechnology Information, PAM - protospacer adjacent motif, PBP 2A - penicillin binding protein 2A, RT-qPCR - reverse transcriptase quantitative real-time polymerase chain reaction, tracr - trans-activating CRISPR, UDG - uracil DNA glycosylase