Barcoded Phage Knockout Profiling for Gene Essentiality Mapping
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Solution Overview
Problem
Current methods for screening essential genes in phage genomes are limited by the lack of scalable tools for gene knockout libraries, especially for obligate lytic phages, which do not integrate into the host genome, and the complexity of anti-phage defense systems in bacteria.
Innovation Solution
A method called PhageMaP uses Cas9-recombinase-mediated homologous recombination to create barcoded phage knockout libraries, inserting DNA barcodes into defined loci of phage genomes, allowing for high-throughput screening of gene essentiality and profiling by quantifying barcode abundances post-challenge.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If transposases or CRISPR-Cas9 are introduced to disrupt genes for gene knockout screens, then gene essentiality can be screened, but the method is not scalable to phage genomes which lack cellular machinery
Solution Approach 1:
The patent uses an intermediary system where a bacterial host cell provides the cellular machinery (Cas nuclease and recombinase) that the phage lacks. The host cell is transformed with a barcoded donor plasmid library, and when infected by phage, the host's cellular machinery mediates the homologous recombination process to insert barcodes into the phage genome, enabling gene knockout screens in phages without requiring the phage to possess cellular machinery itself.
Solution Approach 2:
The patent creates copies of the phage genome with inserted barcodes through homologous recombination. The barcoded donor plasmid library serves as a template that is copied into the phage genome at specific loci defined by gRNA spacer sequences. This copying mechanism allows systematic disruption of phage genes while maintaining the ability to track and screen individual gene functions through the barcode sequences.
2Ease of manufacture
If recombineering tools for bacterial genome engineering are used, then gene manipulation is effective, but these tools are ineffective for obligate lytic phages that do not integrate into the host genome
Solution Approach 1:
The patent segments the gene manipulation process into distinct functional components: (1) gRNA spacer sequences that define target loci in the phage genome, (2) homology arms that guide recombination, (3) barcode inserts that disrupt gene function, and (4) a host cell system that provides the necessary enzymatic machinery. This segmentation allows the tool to be adapted to obligate lytic phages by separating the phage-specific targeting elements from the cellular machinery provided by the host.
Solution Approach 2:
The patent changes the operational parameters of gene manipulation by shifting from phage-autonomous systems to host-mediated systems. Key parameter changes include: using host-expressed Cas nuclease instead of phage-encoded enzymes, using host-provided recombinase activity, and utilizing plasmid-based donor templates rather than phage-integrated systems. These parameter changes enable effective gene manipulation in obligate lytic phages while maintaining high throughput and precision.
3Productivity
If high throughput screening methods are developed for phage genomes, then gene essentiality can be assessed, but the complexity of anti-phage defense systems in bacteria remains a challenge
Solution Approach 1:
The patent creates a universal platform that can screen phage gene essentiality across diverse bacterial hosts and conditions. The barcoded phage variant library can be challenged against multiple bacterial strains simultaneously, and the same screening methodology works regardless of the specific anti-phage defense systems present in the hosts. This multi-functionality allows the system to handle the complexity of various defense systems by treating them as variable parameters rather than fundamental limitations.
Solution Approach 2:
The patent implements feedback through quantitative analysis of barcode abundances before and after bacterial challenge. By sequencing and comparing barcode frequencies in the phage library pre- and post-infection, the system receives feedback on which phage variants survived the anti-phage defense systems. This quantitative feedback allows systematic identification of conditionally essential genes and defense interaction mechanisms, transforming the complexity of defense systems into measurable data for high-throughput analysis.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
PhageMaP enables systematic and high-throughput screening of phage gene essentiality, identifying conditionally essential genes and anti-phage defense interactions, and facilitating effective phage engineering across diverse bacterial hosts and conditions.
Implementation Method 1
double stranded cleavage of the target phage genomes by the Cas nuclease-gRNA
Implementation Method 2
subsequent recombinase-mediated homologous recombination with the donor plasmid inserts the barcodes into the genomic loci in the target phage
Data Source
AI summary
Described herein is the Phage high-throughput approach for gene essentiality Mapping and Profiling (PhageMaP) platform to generate pooled, barcoded phage knockouts for high throughput screening of conditional phage gene essentiality. A barcoded donor plasmid library is prepared wherein each member includes a single guide RNA (gRNA) spacer sequence and 5′ and 3′ homology arm sequences. A host cell engineered to express a Cas nuclease and a recombinase is then transformed with the donor plasmid library followed by infection with a population of target phage to provide a barcoded target phage variant library produced by double stranded cleavage of the target phage genomes by the Cas nuclease-gRNA and subsequent recombinase-mediated homologous recombination with the donor plasmid. Insertion of the barcodes at the genomic loci disrupts the function of the genomic loci and provides the barcoded target phage variant library which may then be isolated.