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

我们利用从纤毛菌基因组中筛选到的CRISPR- cas13a，并在CRISPR区域引入靶向mecA的间隔片段，成功构建了抗MRSA的抗菌衣壳（AB衣壳）。该AB衣壳被证明具有序列特异性杀伤活性。然而，由于CRISPR-Cas13a的大小相对较大（约5kb），我们的下一个挑战是确定合适的位置将它们插入噬菌体基因组而不中断噬菌体组装。到目前为止，我们可以在噬菌体的晚期基因（裂解模块）周围插入CRISPR-Cas13a。我们通过删除噬菌体内溶素，将CRISPR-Cas13a装载到噬菌体中，并用CRISPR-Cas13a代替。用CRISPR-Cas13a替代天然内溶素使我们能够构建一种噬菌体，它可以选择性地杀死携带目标基因的细菌。然而，杀伤活性并不像预期的那么强，这可能是其他因素的存在或CRISPR-Cas13a插入的不利位置。我们用CRISPR-Cas13代替噬菌体内溶素，因为我们想要去除噬菌体的裂解特性，以确保由于CRISPR-Cas13a的裂解活性而发生裂解，因为我们的目的之一是生成一个基于基因的检测系统。不幸的是，当我们使用高滴度的噬菌体时，仍然可以观察到缺乏内溶素的噬菌体的裂解活性，这可能表明存在其他因素影响噬菌体的裂解行为。本项目的后续研究应包括确定在噬菌体基因组中插入CRISPR-Cas13a的其他合适位置。

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.