Development of a novel MRSA genotyping method that does not require nucleic acid manipulation and optical device

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K15652
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 52010:General internal medicine-related
• Research Institution: Jichi Medical University
• Principal Investigator: Tan Xin Ee 自治医科大学, 医学部, 助教 (70828475)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: MRSA genotyping / CRISPR-Cas / Phagemid

Outline of Final Research Achievements

This study aimed to establish a simple and cheap MRSA genotyping system by exploiting the collateral RNA cleavage ability of CRISPR-Cas13a ensuing sequence-specific recognition of target gene. For this purpose, different genetic discriminants commonly used for MRSA genotyping have been selected. Their conserved regions were determined and used as templates for the design of potential crRNA oligonucleotides. Next, crRNAs were loaded onto a plasmid carrying phage packaging site and CRISPR-Cas13a (phagemid). These phagemids were then packaged into our candidate broad-host-range phage, generating a library of ‘typing tools’ targeting different genetic determinants of MRSA. Finally, these CRISPR-Cas13a-based ‘typing tools’ were used to infect S. aureus RN4220 expressing the corresponding target genes. The ability of these capsids to kill the target bacteria sequence-specifically were confirmed. Our study showed that AB-capsids have the potential to be developed as MRSA genotyping tools.

Free Research Field

感染症学・微生物学

Academic Significance and Societal Importance of the Research Achievements

本申請の成果は、ファージとCRISPR-Cas13aシステムを組み合わせた新しいMRSA遺伝子型別法の構築である。本システムは、スペーサー配列を設計するだけで、MRSAの遺伝子型別であるPFGE、SCCmec、MLST、coaおよびspa型別を安価かつ簡便に決定できる新しいMRSA検査法を提供できる。また、遺伝子の増幅を必要とせず、標的遺伝子を高感度に検出できる。さらに、高精度に標的遺伝子を認識できることで再現性にも優れている。特に、crRNA配列とファージの宿主相互作用による特異性により、明確な鑑別と解釈が可能であり、試験結果に差異が生じないことも大きな利点である。