CRISPR-Cas Spacer Acquisition via Exogenous Cas1 Cas2
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Solution Overview
Problem
Non-adapting CRISPR-Cas systems in bacteria, such as those found in Lactococcus cremoris, lack the ability to acquire new spacers, rendering them incapable of adapting to new threats like bacteriophages, which can lead to bacterial culture failures in industrial applications, particularly in the food industry, resulting in economic losses and product quality issues.
Innovation Solution
Introduction of Cas1 and Cas2 genes from Lactococcus raffinolactis into non-adapting CRISPR-Cas systems in Lactococcus cremoris bacteria, enabling the acquisition of new spacers through controlled exposure to target nucleic acids or stressors, thereby adapting the CRISPR-Cas system without altering its fundamental behavior.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If non-adapting CRISPR-Cas systems are used in bacteria, then the system maintains stable and predictable behavior, but the bacteria cannot acquire new spacers and adapt to new threats like bacteriophages
Solution Approach 1:
The patent introduces an exogenous adaptation module comprising Cas1 and Cas2 proteins as intermediaries that bridge the gap between the non-adapting CRISPR-Cas system and the ability to acquire new spacers. These mediator proteins enable the system to incorporate foreign DNA into the CRISPR array without altering the core non-adapting characteristics of the original system.
Solution Approach 2:
The patent combines the non-adapting CRISPR-Cas system with an exogenous adaptation module in a hybrid system. This merging allows the bacteria to retain the stability of the original system while gaining the adaptability to acquire new spacers through the integrated Cas1-Cas2 machinery.
2Adaptability or versatility
If the CRISPR-Cas system is modified to enable adaptation, then the bacteria can acquire immunity against new threats, but the fundamental non-adapting behavior of the system is altered
Solution Approach 1:
The patent segments the CRISPR-Cas system into distinct functional modules: the core non-adapting CRISPR-Cas machinery and the exogenous adaptation module. This segmentation allows the adaptation function to be added as a separate, controllable component that does not fundamentally alter the behavior of the original CRISPR-Cas system.
Solution Approach 2:
The patent introduces dynamic control mechanisms that allow the adaptation module to be activated or deactivated as needed. This enables the system to switch between adapting and non-adapting states, providing flexibility while preserving the fundamental characteristics of the original system when adaptation is not required.
3Adaptability or versatility
If bacteria are exposed to target nucleic acids to induce spacer acquisition, then new spacers are incorporated into the CRISPR array, but the process requires controlled exposure protocols and may not be sufficient without additional components
Solution Approach 1:
The patent introduces the Cas1 and Cas2 proteins in advance as part of the exogenous adaptation module, preparing the system for spacer acquisition before exposure to target nucleic acids. This preliminary provision of essential components simplifies the overall process by ensuring all necessary machinery is in place before adaptation is triggered.
Data Source
AI summary
Provided herein are compositions for enabling natural adaptive spacer acquisition in CRISPR-Cas systems incapable of adaptation, e.g., non-adapting lactococcal CRISPR-Cas systems. Also provided are methods for using the compositions and products thereof to produce bacterial strains that are resistant, such as bacteriophage resistant, and/or evolved to have a desirable phenotype. Non-adapting CRISPR-Cas systems adapted according to the methods provided herein, and bacteria containing such adapted non-adapting CRISPR-Cas systems, are further provided. The compositions and methods provided herein allow for the controlled induction of natural spacer acquisition via adaptation in CRISPR-Cas systems that are fundamentally incapable of adaptation without modifying the features underlying the non-adapting behavior of the CRISPR-Cas system.