Episomal Replicon for Scarless Synthetic DNA Integration
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Solution Overview
Problem
Current methods for introducing synthetic DNA into the E. coli genome are complex, time-consuming, and require the design and cloning of multiple spacer sequences, limiting scalability and efficiency.
Innovation Solution
A method involving a host cell with an episomal replicon containing a donor nucleic acid sequence flanked by homologous recombination sequences, along with helper proteins and an RNA-guided DNA endonuclease, to facilitate excision and recombination of the synthetic DNA into the target nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If REXER method is used to introduce synthetic DNA into E. coli genome, then genome engineering capability is achieved, but the process is complex and time-consuming requiring design and cloning of multiple spacer sequences
Solution Approach 1:
The patent extracts the spacer design and cloning steps from the genome integration process by using a self-excising BAC system. The BAC automatically excises itself from the helper plasmid and integrates into the target genome, eliminating the need for separate spacer sequence design and cloning operations that were required in traditional REXER methods.
Solution Approach 2:
The BAC system performs self-service by autonomously excising from the helper plasmid and integrating into the target genome without requiring external spacer molecules. The integrated cassette contains all necessary elements (origin of replication, selectable markers, and target site) to facilitate self-directed integration, making the system independent of complex spacer design.
2Productivity
If REXER method is used for genome synthesis, then synthetic DNA can be integrated into genome, but the process takes four days from cells to clonal colonies
Solution Approach 1:
The BAC is pre-prepared with all necessary integration elements (origin of replication, selectable markers, and target site homology) before introduction into the cell. This preliminary preparation of the integrated cassette eliminates the need for time-consuming spacer design, synthesis, and cloning steps that would otherwise be required during the integration process.
Solution Approach 2:
The patent merges multiple functions into a single integrated cassette: the origin of replication, selectable markers, and target site homology regions are combined into one BAC structure. This consolidation allows all necessary elements for successful genome integration to be delivered in a single molecular entity, reducing the number of sequential steps required.
3Manufacturing precision
If multiple REXER steps are used to replace entire E. coli genome, then complete genome recoding is achieved, but the process requires 38 steps with bespoke spacer pairs
Solution Approach 1:
The BAC system provides a universal platform for genome recoding that can be applied to any target site without requiring custom spacer design. The standardized integrated cassette with selectable markers and origin of replication can be used repeatedly for different genomic locations, making the recoding process universally applicable across multiple steps.
Solution Approach 2:
The genome recoding process is segmented into discrete BAC-mediated integration events, where each BAC carries a specific recoded segment. This segmentation allows systematic replacement of genomic regions through a standardized protocol, eliminating the need for complex multi-step spacer design while maintaining precision.
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 method simplifies the introduction of synthetic DNA into the E. coli genome, enabling accelerated and scalable genome engineering and synthesis, with the potential for scarless integration of large synthetic DNA fragments.
Implementation Method 1
a spacer array is introduced into the competent cells to activate Cas9 mediated excision of the insert containing the synthetic DNA from the BAC
Implementation Method 2
The precisely excised insert is then used, by the lambda red recombination machinery, to insert the synthetic DNA into the genome in place of the corresponding genomic DNA
Data Source
AI summary
In an aspect, the present invention relates to methods of introducing a sequence of interest into a target nucleic acid. The invention also relates to methods of assembling nucleic acid sequences comprising iterating the methods of introducing a sequence of interest into a target nucleic acid, as well as assembly of replicons encoding larger amounts heterologous nucleic acid.