Optimization of T4 phage engineering via CRISPR/Cas9

A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to pro...

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Bibliographic Details
Published in:Scientific reports 2020-10, Vol.10 (1), p.18229, Article 18229
Main Authors: Duong, Michelle M., Carmody, Caitlin M., Ma, Qinqin, Peters, Joseph E., Nugen, Sam R.
Format: Article
Language:English
Subjects:
631/326/1321
631/326/325
631/326/432
Bacteria - metabolism
Bacteria - virology
Bacteriophage T4 - genetics
Bacteriophage T4 - metabolism
Biological control
CRISPR
CRISPR-Cas Systems - genetics
Editing
Engineering
Gene Editing - methods
Gene Editing - standards
Genetic Engineering - methods
Genetic Engineering - standards
Genome editing
Genomes
Humanities and Social Sciences
multidisciplinary
Phages
Science
Science (multidisciplinary)
Viral Plaque Assay - methods
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Summary:A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03–3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-75426-6