Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7
Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7
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DOI：
10.3390/v12020193
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发表时间：
2020-02-01
期刊：
影响因子：
4.7
通讯作者：
Millard, Andrew
中科院分区：
文献类型：
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作者：
Grigonyte, Aurelija M.;Harrison, Christian;Millard, Andrew
随着最近对使用裂解性噬菌体作为治疗剂的兴趣的增加，迫切需要了解它们的基本生物学以使其基因组的工程化成为可能。目前的噬菌体工程方法依赖于同源重组，然后通过选择系统来识别重组噬菌体。对于噬菌体T7，宿主基因cmk或trxA已被用作选择机制，与I型和II型CRISPR系统一起沿着以针对野生型噬菌体进行选择并富集所需突变体。在这里，我们系统地比较所有三个系统，我们表明，使用基于标记的选择是最有效的方法，我们用它来产生多个T7尾纤维突变体。此外，我们发现II型CRISPR-Cas系统更容易使用，并且在噬菌体T7的工程化中通常比I型系统更有效。这些结果为将来更有效地工程化噬菌体T7提供了基础。
With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.