Liyanagedera, Sahan B. W., Williams, Joshua, Wheatley, Joseph P., Biketova, Alona Yu, Hasan, Muhammad, Sagona, Antonia P., Purdy, Kevin J., Puxty, Richard J., Feher, Tamas and Kulkarni, Vishwesh V. (2022) SpyPhage : a cell-free TXTL platform for rapid engineering of targeted phage therapies. ACS Synthetic Biology, 11 (10). pp. 3330-3342. doi:10.1021/acssynbio.2c00244 ISSN 2161-5063.
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Abstract
The past decade has seen the emergence of multidrug resistant pathogens as a leading cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including host range alterations, constitutive lytic growth, and control over phage replication. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to this problem, we propose the SpyPhage system where a “scaffold” bacteriophage is engineered to incorporate a SpyTag moiety on its capsid head to enable rapid postsynthetic modification of their surfaces with SpyCatcher-fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and whole genome assembly, we targeted a SpyTag capsid fusion to K1F, a phage targeting the pathogenic strain Escherichia coli K1. We demonstrate for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying “scaffold” genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations.
| Item Type: | Journal Article |
|---|---|
| Subjects: | Q Science > QD Chemistry |
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) |
| Library of Congress Subject Headings (LCSH): | Bacteriophages -- Genetic engineering, Bacteriophages -- Therapeutic use, Self-assembly (Chemistry), CRISPR (Genetics) |
| Journal or Publication Title: | ACS Synthetic Biology |
| Publisher: | American Chemical Society |
| ISSN: | 2161-5063 |
| Official Date: | 21 October 2022 |
| Dates: | Date Event 21 October 2022 Published 4 October 2022 Available 2 September 2022 Accepted |
| Volume: | 11 |
| Number: | 10 |
| Page Range: | pp. 3330-3342 |
| DOI: | 10.1021/acssynbio.2c00244 |
| Status: | Peer Reviewed |
| Publication Status: | Published |
| Re-use Statement: | This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acssynbio.2c00244 |
| Access rights to Published version: | Restricted or Subscription Access |
| Date of first compliant deposit: | 13 October 2022 |
| Date of first compliant Open Access: | 4 October 2023 |
| RIOXX Funder/Project Grant: | Project/Grant ID RIOXX Funder Name Funder ID 1917070 [EPSRC] Engineering and Physical Sciences Research Council BB/M017982/1 [BBSRC] Biotechnology and Biological Sciences Research Council BB/N011872/1 [BBSRC] Biotechnology and Biological Sciences Research Council |
| URI: | https://wrap.warwick.ac.uk/170021/ |
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