Episomal Replicon Genome Editing for Large DNA Insertion
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Solution Overview
Problem
Current methods for genome synthesis and manipulation, such as CRISPR-Cas systems, face limitations in efficiently inserting or replacing large DNA sequences within bacterial genomes, particularly in E. coli, due to low transformation efficiency and off-target mutations, which hinders the ability to introduce significant changes or recode genomes effectively.
Innovation Solution
A method involving an episomal replicon with a donor nucleic acid sequence and helper proteins like CRISPR/Cas9 for single-step insertion or replacement of up to 100 kb of synthetic DNA into the E. coli genome, utilizing double-stranded breaks and simultaneous selection for marker gain and loss to ensure precise recombination and minimize off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas systems are used for genome editing, then site-directed mutagenesis can be achieved, but transformation efficiency decreases and off-target mutations increase when large DNA sequences are inserted or replaced
Solution Approach 1:
The genome editing process is divided into two distinct phases: first, creating double-stranded breaks at target sites using CRISPR-Cas9 to enable precise mutagenesis; second, using an episomal replicon system to deliver and integrate large donor DNA sequences. This segmentation allows each step to be optimized independently, maintaining precision while improving transformation efficiency for large inserts.
Solution Approach 2:
An episomal replicon serves as an intermediary carrier to deliver large donor DNA sequences into the bacterial cell. The replicon temporarily maintains the donor DNA in a replicated state, allowing efficient recombination with the target genome without requiring direct transformation of large linear DNA fragments, thus overcoming the transformation efficiency limitation.
2Productivity
If iterative site directed mutagenesis is used to edit multiple codons, then genome recoding can be achieved, but the number of off-target mutations increases
Solution Approach 1:
Multiple codon alterations are designed and prepared in advance within a single donor DNA sequence on the episomal replicon. This preliminary assembly of all desired mutations in one construct allows them to be introduced simultaneously through a single recombination event, eliminating the need for iterative mutagenesis steps that would accumulate off-target mutations.
Solution Approach 2:
Multiple separate mutation operations are merged into a single genome replacement event. By combining all desired codon changes into one donor DNA sequence that is introduced via the episomal replicon system, the process achieves high productivity while minimizing off-target effects, as the single recombination event replaces the entire target region in one step.
3Quantity of substance
If linear DNA transformation is used for large inserts, then genome insertion can be achieved, but transformation efficiency becomes severely restricted
Solution Approach 1:
The episomal replicon acts as an intermediary vehicle that can accommodate and maintain large donor DNA sequences (up to 100 kb) in a replicated state within the bacterial cell. This intermediary system bypasses the severe transformation efficiency restrictions of direct linear DNA uptake, allowing large inserts to be introduced efficiently through the replicon's replication and segregation mechanism followed by recombination.
Solution Approach 2:
The system changes the physical state and delivery mechanism of the donor DNA. Instead of transforming large linear DNA fragments directly (which has severe efficiency restrictions), the donor DNA is delivered as part of an episomal replicon that can be maintained in a circular, replicated state, fundamentally changing the transformation parameters and enabling efficient introduction of large inserts.
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
Enables efficient, length-independent insertion or replacement of large DNA sequences with high specificity, allowing for extensive genome recoding and synthesis, overcoming previous limitations in transformation efficiency and off-target issues.
Implementation Method 1
inducing excision of said donor nucleic acid sequence... inducing at least one double stranded break in the target nucleic acid sequence
Data Source
AI summary
The invention relates to a method comprising a) providing a host cell said host cell comprising an episomal replicon, said episomal replicon comprising a donor nucleic acid sequence, said host cell further comprising a target nucleic acid, b) providing helper protein(s) capable of supporting nucleic acid recombination in said host cell c) providing helper protein(s) and/or RNAs capable of supporting nucleic acid excision in said host cell wherein said donor nucleic acid sequence comprises in order. 5′-homologous recombination sequence 1-sequence of interest-homologous recombination sequence 2-3′ wherein said sequence of interest comprises a positive selectable marker d) inducing excision of said donor nucleic acid sequence e) incubating to allow recombination between the excised donor nucleic acid and said target nucleic acid f) selecting for recombinants having incorporated said donor nucleic acid into said target nucleic acid. Also described are nucleic acids and cells.