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

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K15652
• Research Institution: Jichi Medical University
• Principal Investigator: タン シンイー 自治医科大学, 医学部, ポスト・ドクター (70828475)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: MRSA genotyping / CRISPR-Cas / Phagemid

Outline of Annual 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. Genomic sequences of these genes of interest among different clinical S. aureus strains were aligned and the conserved regions were determined. These regions were then used as templates for the design of potential crRNA oligonucleotides. Following that, the ability of CRISPR-Cas13a to specifically recognize the designed crRNA oligonucleotides were evaluated by sequentially transforming S. aureus RN4220 with expression plasmids carrying target gene and programmed CRISPR-Cas13a. Optimization of crRNA sequences are still in progress.

Next, it is necessary to identify phage with broad host range in order to deliver the programmed CRISPR-Cas13a to a vast array of S. aureus strains. We have recovered a candidate temperate phage through chemical induction of in-house clinical S. aureus strains using Mitomycin C. This particular phage was found to be able to infect diverse S. aureus strains, reaching a percent lytic activity of more than 95. For the ease of construction, the custom-synthesized crRNA oligonucleotides have been loaded onto a plasmid carrying phage packaging site pac and CRISPR-Cas13a. In the R3 research year, we have successfully packaged CRISPR-Cas13a carrying crRNA oligonucleotides into our candidate phage using this alternative approach.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

According to the proposed research timeline, we have completed identification of candidate genes for MRSA genotyping and isolation of broad-host-range phage in the R3 research year. Optimization of crRNA sequences is also ongoing as planned.

Strategy for Future Research Activity

In the following research year, optimization of all candidate crRNA oligonucleotides will be continued to finalize the sequences that possess high variation detection ability among different genotypes of S. aureus. After determining the ideal crRNA oligonucleotides, corresponding phagemids will be packaged into the broad-host-range phage identified beforehand using the established phagemid approach to generate a library of ‘typing tools’ targeting different genetic determinants. Finally, the engineered chimeric phages will be used to infect various clinical strains of our culture collection as well as standard strains to evaluate their efficiencies and applicability for MRSA genotyping.