<br>通过 V 型 CRISPR-Cas 基因组编辑进行体内展示的工程 T4 噬菌体,ACS Synthetic Biology

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通过 V 型 CRISPR-Cas 基因组编辑进行体内展示的工程 T4 噬菌体
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-09-21 , DOI: 10.1021/acssynbio.1c00251
Junhua Dong _{1,

2,

3} , Cen Chen _{1,

2,

3} , Yuepeng Liu _{1,

2,

3} , Jingen Zhu ₄ , Mengling Li _{1,

2,

3} , Venigalla B Rao ₄ , Pan Tao _{1,

2,

3}

Affiliation

噬菌体 T4 具有大尺寸、衣壳结构以及蛋白质和 DNA 递送的高有效载荷能力，因此在生物医学应用方面具有巨大潜力。然而，对其基因组进行基因工程改造并受胞嘧啶羟甲基化和葡萄糖基化严重修饰并不容易。噬菌体的葡萄糖基羟甲基胞嘧啶（ghmC）基因组对大多数限制性核酸内切酶完全耐药，并且对 CRISPR-Cas 系统表现出不同程度的耐药性。在这里，我们发现 V 型 CRISPR-Cas12a 系统与 II 型 CRISPR-Cas9 系统相比，它显示出 ghmC 修饰基因组的有效切割，可以协同用于生成重组 T4 噬菌体。专注于表面展示，我们分析了噬菌体 T4 外衣壳蛋白 Hoc（高抗原性外衣壳蛋白）和 Soc（小外衣壳蛋白）在体内将外源肽和蛋白质拴在 T4 衣壳上的能力。我们的数据表明，虽然这些肽可以在噬菌体感染期间成功表达和显示，但较短的肽的拷贝数比全长蛋白高得多。然而，通过使用强 T7 RNA 聚合酶表达系统驱动转基因的表达，可以提高后者的拷贝数。这种受 CRISPR 启发的方法有可能将噬菌体的应用扩展到各种基础和转化研究项目。

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Engineering T4 Bacteriophage for In Vivo Display by Type V CRISPR-Cas Genome Editing

Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether, in vivo, foreign peptides and proteins to T4 capsid. Our data show that while these could be successfully expressed and displayed during the phage infection, shorter peptides are present at a much higher copy number than full-length proteins. However, the copy number of the latter could be elevated by driving the expression of the transgene using the strong T7 RNA polymerase expression system. This CRISPR-inspired approach has the potential to expand the application of phages to various basic and translational research projects.

更新日期：2021-10-15

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