ORACLE Phage Library Method for Unbiased Variant Screening
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Solution Overview
Problem
Current methods for creating phage variants, such as bacteriophages, are laborious, difficult to interpret, and not suitable for high-throughput screening, often resulting in biased populations and making it challenging to deconvolve the individual effects of mutations, which hampers phage research and application in areas like biocontrol and antibiotic-resistant bacteria treatment.
Innovation Solution
The ORACLE method (Optimized Recombination, Accumulation, and Library Expression) is introduced, which involves preparing acceptor phages with recombination sites, inserting mutated target genes, selectively accumulating recombined phages using a counterselection system, and expressing the library to produce large, unbiased phage libraries, enabling rapid screening of phage mutants and broad sampling of natural phage populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional random mutagenesis screening is used, then phage variants can be generated, but the process is slow and generates many variants with multiple mutations making it difficult to deconvolve individual mutation effects
Solution Approach 1:
The patent segments the mutagenesis process by using a library of gRNAs that each target specific single nucleotide positions in the phage genome. This allows systematic generation of single-mutation variants rather than random multi-mutation variants, enabling clear deconvolution of individual mutation effects while maintaining high throughput screening capability
Solution Approach 2:
The patent introduces CRISPR-Cas9 system as an intermediary mechanism to achieve precise single-nucleotide mutagenesis. The gRNA-Cas9 complex mediates targeted cleavage at specific positions, and the repair process introduces controlled single mutations. This intermediary system enables precise mutagenesis while maintaining high throughput, resolving the contradiction between screening speed and mutation effect deconvolution
2Productivity
If directed evolution is used, then phage variants can be enriched, but only a small group of variants is enriched that are biased towards variants growing better on the host used to create the phages
Solution Approach 1:
The patent performs preliminary action by creating a comprehensive library of single-nucleotide variants across the entire target gene before any selection pressure is applied. This ensures all possible single-mutation variants are represented in the library prior to enrichment, preventing bias towards pre-existing better-growing variants and enabling discovery of variants with diverse growth characteristics
Solution Approach 2:
The patent changes the parameter of variant diversity by using a counterselection system that enriches for variants with altered host range or reduced fitness on the original host. This parameter change allows enrichment of variants that would be depleted in traditional directed evolution, reducing library bias and increasing adaptability of the phage population
3Manufacturing precision
If CRISPR-Cas9 counterselection is used, then recombined phages can be selectively accumulated, but the process requires multiple host bacteria transformations
Solution Approach 1:
The patent merges multiple functions into a single host bacteria strain. The host simultaneously provides: (1) the target gene for phage infection, (2) the CRISPR-Cas9 counterselection system for selective accumulation, and (3) the recombination machinery for phage genome integration. This merging reduces the number of separate transformation steps while maintaining high purity of the final phage library
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
ORACLE enables the production of large, unbiased phage libraries, allowing for comprehensive evaluation of host range, phage stability, and characterization of metagenomics parts, facilitating the discovery of gain-of-function variants and overcoming the limitations of traditional methods by minimizing library bias and enabling deep mutational scanning without preference for better-growing phages.
Implementation Method 1
recombining a mutated target gene into a portion of the population of acceptor phages by replicating the population of acceptor phages within a first host bacteria, the first host bacteria comprising a first helper plasmid and a recombination plasmid, wherein the recombining produces a mixed phage population comprising acceptor phages and recombined phages
Implementation Method 2
selectively accumulating the recombined phages from the mixed phage population by replicating the mixed phage population within a second host bacteria, the second host bacteria comprising a second helper plasmid and a counterselection system, wherein the counterselection system suppresses replication of the acceptor phages
Data Source
AI summary
Described herein is a method of preparing an unbiased library of phage variants, comprising (a) preparing a population of “acceptor phage”; (b) removing an endogenous target gene and inserting gene variants into the acceptor phage genomes; (c) enriching the recombined phages; and (d) expressing the library for selection. The acceptor phage is a lytic phage comprising a synthetic genome wherein the target gene of interest is flanked by recombinase sites. The acceptor phage infects a first host bacteria expressing a recombination plasmid facilitating recombination. The phages then infect a second host bacteria expressing a counterselection system that accumulates recombined phage variants and selecting against non-recombined phages. The accumulated phage variants infect a third host bacteria. The phage library may then be sequenced and characterized.