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

我们成功地构建了一个抗菌衣壳（AB衣壳），通过使用CRISPR-Cas 13 a，这是从Leptotrichia的基因组中筛选，并引入一个间隔区，靶向mecA到CRISPR区域。这种AB衣壳被证明具有序列特异性杀伤活性。然而，由于CRISPR-Cas 13 a的相对较大的尺寸（约5 Kb），我们的下一个挑战是确定合适的位置以将它们插入噬菌体基因组中而不中断噬菌体组装。到目前为止，我们可以设法将CRISPR-Cas 13 a插入噬菌体的晚期基因（裂解模块）周围。我们通过删除噬菌体内溶素将CRISPR-Cas 13 a加载到噬菌体中，并用CRISPR-Cas 13 a替换它。用CRISPR-Cas 13 a替换天然内溶素使我们能够构建选择性杀死携带靶基因的细菌的噬菌体。然而，杀伤活性不如预期的强，这表明存在其他因素或CRISPR-Cas 13 a插入的不利位置。我们用CRISPR-Cas 13替换噬菌体内溶素，因为我们想要去除噬菌体的裂解特性，以确保由于CRISPR-Cas 13 a的切割活性而发生裂解，因为我们的目的之一是产生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，仍然可以观察到内溶素缺陷型噬菌体的裂解活性，这可能表明存在影响噬菌体裂解行为的其他因素。该项目的后续研究应包括确定在噬菌体基因组中插入CRISPR-Cas 13 a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.