Abstract
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.
Highlights
A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform
Our results indicate that the rate limiting factor in T4 phage engineering using CRISPR/Cas[9] is crRNA selection; effective crRNAs can overcome barriers otherwise imposed by the DNA modifications in T4 phages
To improve the efficiency of employing the CRISPR/Cas[9] system to bioengineer T4 phages we reviewed and evaluated the pre-existing plasmids targeted for this system
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. Other innovative approaches include using type I-E CRISPR/Cas[30] as a counter selection tool and more recently, whole phage genome reconstruction in vitro or within yeast[31,32]. While these techniques have certain useful applications, there are issues that commonly arise when they are used to engineer large DNA genomes due to the existence of modified bases, errors during the recombination of targeted regions, and the overall impurity of samples when isolating from in vitro systems. This group observed that CRISPR/Cas[9] is ineffective at cleaving the cytosine hydroxylmethylated and glucosylated modified T4 genome; this contradicts an earlier finding by Church et al.[27], highlighting a need to further evaluate the role of CRISPR/Cas[9] in editing phages with modified bases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
