Bacteriophage Genome Engineering via CRISPR Cleavage and Recombination
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Solution Overview
Problem
Current methods for engineering bacteriophage genomes are inefficient due to the compact nature of phage genomes, limited restriction sites, and toxicity to bacterial hosts, making it challenging to integrate heterologous nucleic acid sequences and maintain stability of large engineered genomes.
Innovation Solution
A method involving the use of sgRNA-CRISPR enzyme complexes to cleave phage genomes and recombine them with heterologous nucleic acid sequences, utilizing a recombination system to produce recombinant bacteriophages that express bioluminescent, fluorescent, or chemiluminescent proteins, and employing CRISPR systems to enrich for recombinant genomes while blocking wild-type phage replication.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional cloning techniques are used to engineer phage genomes, then the process is simple and familiar, but the techniques are inefficient and challenging to scale up due to compact genomes with few restriction sites
Solution Approach 1:
The patent replaces traditional mechanical cloning techniques (restriction enzymes, ligases, transformation) with an in vivo recombination system using bacteriophage P1 or P22-mediated transduction. This substitution enables efficient genome integration by utilizing the natural recombination machinery of the bacterial host and phage vectors, overcoming the limitations of few restriction sites and compact genome structure.
Solution Approach 2:
The patent introduces a bacterial host cell as an intermediary system that facilitates phage genome engineering. The host cell provides the recombination machinery and metabolic pathways necessary for integrating heterologous DNA into the phage genome, enabling efficient manipulation that would be difficult to achieve through direct in vitro methods alone.
2Reliability
If in vitro engineering strategies are used to create phage variations, then the process is controllable, but the methods are generally inefficient and challenging to scale up
Solution Approach 1:
The patent performs preliminary preparation of the bacterial host cell by introducing the desired heterologous nucleic acid sequence and necessary recombination enzymes before phage infection. This preliminary setup ensures that when the phage infects the host, the recombination events occur efficiently and controllably, maintaining reliability while enabling scalable production through bacterial culture amplification.
Solution Approach 2:
The patent utilizes the bacterial host cell's own recombination machinery and metabolic pathways to perform the genome engineering. The host cell serves itself by providing the enzymatic activities and cellular processes needed for DNA integration, replication, and phage assembly, eliminating the need for complex external intervention and enabling scalable production.
3Stability of the object's composition
If engineering is performed within bacteria to maintain large genomes, then the genomes can be maintained, but phage toxicity to bacteria and stability issues arise
Solution Approach 1:
The patent employs periodic induction of the recombination system using tetracycline or arabinose at specific stages of the infection cycle. This periodic control allows the engineered genome to be assembled and stabilized during specific windows, then the inducing agent is removed to prevent continuous recombination that could generate toxic intermediates or unstable genomes, thereby reducing harm to the bacterial host.
Solution Approach 2:
The patent incorporates selectable markers and screening mechanisms that provide feedback on successful recombination events. This feedback system allows identification and selection of stable recombinant phage genomes while eliminating unstable or toxic variants, maintaining genome stability and reducing harmful effects on the bacterial host through selective pressure.
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 approach allows for higher recovery of recombinant bacteriophage genomes with desired phenotypic properties, overcoming the limitations of traditional phage engineering by enabling efficient integration and expression of heterologous sequences, and enriching for recombinant forms to enhance their abundance.
Implementation Method 1
contacting a first bacteriophage DNA genome with a first sgRNA-CRISPR enzyme complex and a second sgRNA-CRISPR enzyme complex in vivo under conditions where (i) the first sgRNA-CRISPR enzyme complex cleaves a first protospacer sequence within the first bacteriophage DNA genome; and (ii) the second sgRNA-CRISPR enzyme complex cleaves a second protospacer sequence within the first bacteriophage DNA genome
Implementation Method 2
recombining in vivo the cleaved first bacteriophage DNA genome with a heterologous nucleic acid sequence in the presence of a recombination system under conditions to produce the recombinant bacteriophage DNA genome
Implementation Method 3
the heterologous nucleic acid sequence comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof
Implementation Method 4
the heterologous nucleic acid sequence comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof
Implementation Method 5
the heterologous nucleic acid sequence comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof
Data Source
AI summary
The present disclosure provides methods and kits for generating recombinant bacteriophage genomes. Specifically, the present technology provides methods of integrating a heterologous nucleic acid sequence into a bacteriophage DNA genome, and isolating recombinant bacteriophages that express the heterologous nucleic acid sequence.