| 研究実績の概要 |
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 selected target genes were aligned among different S. aureus strains and the conserved regions were used as templates for the design of potential crRNA oligonucleotides.
Next, phages with broad-host-range were isolated as vectors to deliver the programmed CRISPR-Cas13a into various S. aureus strains. We have recovered a candidate temperate phage through Mitomycin C induction of in-house clinical S. aureus strains. 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, custom-synthesized crRNA oligonucleotides have been loaded onto a plasmid carrying phage packaging site pac and CRISPR-Cas13a (phagemids). In the R3 research year, we have successfully packaged those phagemids into our candidate phage and generated a library of ‘typing tools’ targeting different genetic determinants of MRSA.
In the R4 research year, CRISPR-Cas13a-based ‘typing tools’ were used to infect S. aureus RN4220 expressing the corresponding target genes. The ability of these capsids to recognize specific sequences and kill the target bacteria were confirmed. Our study showed that CRISPR-Cas13a-based capsids have the potential to be developed as MRSA genotyping tools.
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