CRISPR Genome Modulation in Vibrio natriegens
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Solution Overview
Problem
Current methods for genome modulation in organisms like Vibrio natriegens are limited by the lack of efficient recombinases and recombination tools, making precise genetic engineering and modification challenging.
Innovation Solution
The use of beta-like recombinases, such as s065, and additional recombination assisting proteins like exonuclease s066 and host nuclease inhibitor gam, along with guide RNAs and Cas proteins, enables targeted nucleic acid sequence modification and recombination in Vibrio natriegens cells, facilitating gene editing and regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR system is used for genome modulation in V. natriegens, then genetic engineering precision is improved, but the complexity of the system increases due to multiple components required
Solution Approach 1:
The CRISPR system is divided into separate functional modules: Cas protein for nuclease activity, guide RNA for target recognition, donor nucleic acid for homology-directed repair, and various assisting proteins. This segmentation allows each component to be optimized independently and introduced into the cell separately, managing complexity while maintaining precision.
Solution Approach 2:
Additional intermediary proteins are introduced to facilitate the interaction between CRISPR components and host machinery. These include exonuclease s066 for processing DNA ends, host nuclease inhibitor gam to protect donor DNA from degradation, and single-strand DNA binding protein s064 to stabilize intermediates during recombination.
2Productivity
If multiple recombination assisting proteins are provided, then recombination efficiency is improved, but the complexity of genetic modification process increases
Solution Approach 1:
Multiple recombination assisting proteins (exonuclease s066, host nuclease inhibitor gam, single-strand DNA binding protein s064) are combined into a coordinated system that works together with the CRISPR components. These proteins are provided together as a package to achieve efficient recombination, merging their functions to overcome individual limitations.
Solution Approach 2:
The recombination assisting proteins perform preliminary actions on the donor nucleic acid before integration: exonuclease s066 processes DNA ends to create appropriate structures, host nuclease inhibitor gam pre-protects donor DNA from host degradation, and single-strand DNA binding protein s064 pre-stabilizes single-stranded intermediates. This preliminary processing ensures efficient subsequent recombination.
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 precise and efficient alteration of target nucleic acid sequences, improved growth rates, and optimized metabolic pathways in Vibrio natriegens, demonstrating enhanced genetic engineering capabilities.
Implementation Method 1
a functioning beta-like recombinase and a donor nucleic acid sequence, wherein the donor nucleic acid sequence is inserted into the target nucleic acid sequence as a result of the functioning beta-recombinase
Implementation Method 2
additional recombination assisting proteins are provided to the cell including the exonuclease s066
Implementation Method 3
a host nuclease inhibitor such as gam
Implementation Method 4
a single-strand DNA binding (SSB) protein s064
Data Source
AI summary
Methods and compositions are provided for modulating expression of a target nucleic acid sequence within a non-E. coli cell. The method includes providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, and providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and wherein the Cas protein modulate the expression of the target nucleic acid sequence.