Development of a novel agent which selectively kills drug-resistant Staphylococcus aureus

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We succeed to construct an antibacterial capsid (AB capsid) against MRSA by using CRISPR-Cas13a which was screened from genome of Leptotrichia together with introducing a spacer that targets mecA into CRISPR region. This AB capsid was proved to have a sequence-specific killing activity. However, due to a relatively large size of CRISPR-Cas13a (approximately 5 Kb), our next challenge was to determine suitable location to insert them into phage genome without interrupting phage assembly. So far, we could manage to insert CRISPR-Cas13a around the late genes (lytic module) of the phage. We loaded the CRISPR-Cas13a into phage by deleting the phage endolysin and replaced it with CRISPR-Cas13a. The replacement of native endolysin with CRISPR-Cas13a enabled us to construct a phage that selectively kills bacteria harboring target genes. However, the killing activity was not strong as expected, suggesting either the presence of other factors or unfavorable locations of CRISPR-Cas13a insertion. We replaced phage endolysin with CRISPR-Cas13 because we wanted to remove lytic property of the phage to ensure that the lysis was occurred because of CRISPR-Cas13a’s cleavage activity, since one of our purpose was to generate a gene-based detection system. Unfortunately, lytic activity of endolysin-deficient phage was still observable when we used high titer of phage which may indicate the presence of other factor influencing phage lytic behavior. The follow up study of this project should include determination of other suitable location to insert CRISPR-Cas13a in the phage genome.