Phage-Resistant Streptococcus via CRISPR Inactivation
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Solution Overview
Problem
Streptococcus thermophilus, a bacterium used in dairy products, faces economic losses due to consistent predation by bacteriophages, with existing CRISPR systems being unstable and prone to evasion by phages with single nucleotide polymorphisms, necessitating a more stable phage resistance mechanism.
Innovation Solution
The method involves inactivating the CRISPR resistance mechanism in Streptococcus thermophilus using antisense RNA constructs to silence cas genes, followed by exposure to bacteriophages to select for bacteriophage-insensitive mutants (BIMs) with alternative resistance mechanisms, ensuring phage resistance independent of CRISPR systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the CRISPR resistance mechanism is used to protect Streptococcus thermophilus against bacteriophages, then phage resistance is provided, but the resistance is unstable and can be evaded by phages with single nucleotide polymorphisms
Solution Approach 1:
The patent extracts and removes the CRISPR resistance mechanism from the bacterial system by inactivating cas genes through antisense RNA constructs. This extraction eliminates the unstable CRISPR-based resistance, forcing the bacteria to develop alternative resistance mechanisms that are more stable and less prone to phage evasion through single nucleotide changes
Solution Approach 2:
Instead of enhancing or activating the CRISPR system to improve resistance, the patent inverts the approach by deliberately inactivating the CRISPR mechanism. This inversion removes the source of instability and allows selection of mutants with alternative, more stable resistance mechanisms that do not rely on CRISPR
2Reliability
If antisense RNA constructs are used to inactivate CRISPR cas genes, then stable non-CRISPR resistance mechanisms are selected, but the process requires additional genetic manipulation steps
Solution Approach 1:
The patent applies preliminary action by first inactivating the CRISPR cas genes using antisense RNA constructs before exposing the bacteria to bacteriophages. This pre-inactivation creates a controlled environment where only non-CRISPR resistance mechanisms can be selected, simplifying the subsequent selection process and ensuring stability
Solution Approach 2:
The antisense RNA constructs serve as an intermediary mechanism to mediate the inactivation of cas genes. Rather than directly modifying the bacterial genome, the antisense RNA provides a temporary, controllable method to suppress CRISPR function, allowing selection of stable mutants without permanent genetic alteration
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 generates BIMs with stable phage resistance through non-CRISPR mechanisms, reducing the risk of phage evasion and enhancing resistance robustness, providing a 'double hurdle' defense against multiple phages.
Implementation Method 1
inactivating the CRISPR resistance mechanism of the parent strain using antisense RNA constructs to silence cas genes
Data Source
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AI summary
The present invention provides a method for the construction of a bacteriophage-insensitive mutant of a Streptococcus thermophilus parent strain whereby the bacteriophage insensitivity is not based on the CRISPR resistance mechanism but based on another mechanism. The method provides a protocol by which this can be achieved by inactivating one or more of the CRISPR systems present in a given strain. The invention also provides the bacteriophage-insensitive mutants as well as their use in a process for making a dairy product.