Circularized Phage DNA Engineering via CRISPR Cleavage
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Solution Overview
Problem
Current methods for engineering bacteriophage genomes are inefficient due to limited restriction sites, compact genomes, and toxicity issues, making it challenging to integrate heterologous nucleic acids and maintain stability, especially when scaling up or engineering phages within bacteria.
Innovation Solution
A method involving CRISPR enzymes to cleave bacteriophage DNA genomes, recombine with heterologous nucleic acids, and enrich the recombinant genomes using exonucleases, allowing for the integration and propagation of recombinant bacteriophages in non-natural hosts, enhancing the recovery of recombinant genomes that express the heterologous nucleic acid sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional cloning techniques are used to engineer phage genomes, then the process is straightforward with standard tools, but the techniques are challenging to scale up and inefficient
Solution Approach 1:
The patent uses circularized intermediate DNA molecules as a mediator in the recombination process. Linear phage genomes are first circularized in vitro to form intermediate structures, which then serve as templates for efficient recombination with heterologous nucleic acids. This circularized intermediate approach bridges the gap between traditional cloning and modern recombination methods, enabling both ease of manipulation and high efficiency.
2Adaptability or versatility
If phage engineering is performed within bacteria, then integration of heterologous nucleic acid can occur, but toxicity of phages to bacteria and stability issues arise
Solution Approach 1:
The patent extracts the recombination process from the bacterial host environment and performs it in vitro. By taking out the engineering step from within bacteria to an extracellular test tube environment, the method avoids phage toxicity to bacteria and genomic instability issues. The circularized intermediate recombination occurs outside the host, then the successful recombinants are introduced back into bacteria for propagation.
3Productivity
If in vitro recombination strategies are used, then scaling up is possible, but the processes are inefficient and challenging to scale up
Solution Approach 1:
The patent segments the phage engineering process into distinct modular steps: (1) in vitro circularization of linear phage genomes to form intermediates, (2) in vitro recombination with heterologous nucleic acids, and (3) introduction into bacterial hosts for propagation. This segmentation allows each step to be optimized independently and performed in parallel, enabling easy scaling while maintaining procedural simplicity.
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
This method significantly increases the recovery of recombinant bacteriophage genomes that express the desired phenotypic properties, demonstrating superior efficiency compared to existing techniques like Break and Recombine 3.0, with an 81% yield of recombinant isolates, compared to 0% using BAR 3.0.
Implementation Method 1
cleaving a first site at the 5′ end of a plurality of linear bacteriophage DNA genomes and a second site at the 3′ end of the plurality of linear bacteriophage DNA genomes with a CRISPR enzyme in vitro
Implementation Method 2
enriching the plurality of circularized bacteriophage DNA genomes by incubating the sample with at least one exonuclease
Data Source
AI summary
The present disclosure provides methods of generating recombinant bacteriophage genomes. Specifically, the present technology provides methods of integrating a heterologous nucleic acid sequence into a linear bacteriophage DNA genome, and isolating recombinant bacteriophages that express the heterologous nucleic acid sequence.