Phage DNA Methylation for Restriction-Resistant CRISPR Delivery
Solution Overview
Problem
Existing bacteriophages face limitations in delivering synthetic DNA due to bacterial restriction-modification systems that cleave foreign DNA with mismatched methylation patterns, limiting their applicability across diverse bacterial strains.
Innovation Solution
Modify the methylation pattern of bacteriophage and phagemid DNA by disrupting endogenous restriction enzymes or introducing heterologous methyltransferases in the production host bacterium, enabling the production of bacteriophages with modified methylation patterns compatible with target bacteria, thereby enhancing DNA delivery efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bacteriophages are used to deliver synthetic DNA to bacteria, then DNA delivery can be achieved, but bacterial restriction-modification systems cleave foreign DNA with mismatched methylation patterns, reducing delivery efficiency
Solution Approach 1:
The patent applies preliminary action by pre-modifying the methylation pattern of bacteriophage DNA during phage production in a host bacterium with altered methylating activity. This advance modification ensures the DNA is protected from restriction enzymes before delivery occurs, resolving the contradiction between delivery efficiency and DNA survival reliability.
Solution Approach 2:
The patent changes the chemical parameter of DNA methylation by using host bacteria with disrupted endogenous restriction-modification systems or introduced heterologous methyltransferases. This parameter change in methylation pattern allows the bacteriophage DNA to evade recognition and cleavage by restriction enzymes in target bacteria, thereby improving both delivery efficiency and DNA survival.
2Adaptability or versatility
If endogenous restriction modification systems are disrupted or heterologous methyltransferases are introduced in production host, then methylation barriers are overcome, but production host complexity increases
Solution Approach 1:
The patent applies the taking out principle by removing or disrupting the endogenous restriction-modification systems in the production host bacterium. This extraction of the problematic R-M system simplifies the production process by eliminating the barrier to DNA methylation modification, while still allowing the introduction of specific heterologous methyltransferases needed for the desired methylation pattern.
Solution Approach 2:
The patent uses heterologous methyltransferases as intermediaries to transfer the desired methylation pattern to the bacteriophage DNA. These intermediary enzymes mediate between the production host and the target bacteria's methylation requirements, enabling compatibility without requiring complex direct modification of the production host's entire methylation system.
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
Enhances the efficiency of introducing heterologous nucleic acids, including CRISPR-Cas systems, into target bacteria by overcoming methylation barriers, allowing selective killing of specific bacterial species.
Implementation Method 1
altering methylating activity of a production host bacterium... introducing into a production host bacterium a polynucleotide encoding at least one heterologous methyltransferase, thereby producing a modified production host bacterium expressing the heterologous methyltransferase; infecting the modified production host bacterium... thereby methylating said bacteriophage DNA
Implementation Method 2
bacterial restriction-modification systems that cleave foreign DNA with mismatched methylation patterns... disrupting the activity at least one enzyme of an endogenous restriction modification system (R-M system)
Data Source
AI summary
The invention relates to the methods for modifying the methylation pattern of bacteriophage DNA and phagemid DNA and to methods for selective killing of bacteria using lysogenic bacteriophages comprising bacteriophage DNA or phagemid DNA comprising components of an engineered CRISPR-Cas system.