Circular Polynucleotide Templates for E. coli Gene Editing
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Solution Overview
Problem
Current methods for editing nucleotide sequences in the genome of Escherichia coli are not efficient and effective, despite advancements in recombinant DNA technology and the use of CRISPR systems.
Innovation Solution
The use of a guide RNA/Cas endonuclease system in combination with a circular polynucleotide modification template to facilitate homologous recombination-mediated gene editing, allowing for precise editing of target sites within the E. coli genome, including gene knock-in and knock-out applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional homologous recombination methods are used for gene editing in E. coli, then the method is applicable across a wide array of organisms, but the editing efficiency is low
Solution Approach 1:
The patent introduces a CRISPR-Cas9 system as an intermediary mechanism to enhance homologous recombination. The Cas9 endonuclease, guided by CRISPR RNA, creates targeted double-strand breaks in the DNA, which then stimulates efficient homologous recombination when a modification template is provided. This intermediary system bridges the gap between traditional HR methods and precise gene editing requirements.
Solution Approach 2:
The patent changes the physical state of the modification template by using a circular polynucleotide structure instead of linear DNA. This circular configuration increases the stability and persistence of the template in the cell, allowing it to remain available for homologous recombination events over an extended period, thereby improving editing efficiency without compromising precision.
2Productivity
If CRISPR-Cas9 system is used to introduce double-strand breaks, then homologous recombination is stimulated, but the complexity of the system increases
Solution Approach 1:
The patent employs a universal CRISPR-Cas9 platform that can be applied to edit any gene target in E. coli by simply changing the CRISPR RNA guide sequence. This multi-functional system maintains a core consistent mechanism (Cas9 endonuclease) while allowing flexible targeting, thereby managing complexity through standardization rather than creating multiple different systems for different applications.
Solution Approach 2:
The patent extracts and utilizes only the essential double-strand break creation function of the CRISPR-Cas9 system, separating this from other complex CRISPR functions. By focusing specifically on Cas9's endonuclease activity to create DSBs that stimulate HR, the system simplifies the overall approach while maintaining high productivity for gene editing applications.
3Ease of manufacture
If linear polynucleotide templates are used for gene editing, then the template can be easily introduced, but the template is quickly degraded and lost in the cell
Solution Approach 1:
The patent transforms the linear polynucleotide template into a circular structure. This curvature provides topological stability, preventing the template from being degraded by exonucleases that act on linear DNA ends. The circular configuration allows the template to persist in the cell for extended periods, maintaining its availability for homologous recombination events without compromising the ease of its initial introduction.
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 provides a robust and efficient method for editing nucleotide sequences in E. coli, enabling precise modifications to various genomic regions, such as promoter sequences, coding sequences, and prophages, with high specificity and efficiency.
Implementation Method 1
The guide RNA component can be designed such that Cas9 recognizes and cleaves DNA containing (i) sequence complementary to a portion of the RNA component
Implementation Method 2
Cas9 recognizes and cleaves DNA containing sequence complementary to a portion of the RNA component
Implementation Method 3
homologous recombination-mediated gene editing at a target site in the genome
Data Source
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AI summary
Compositions and methods are provided for genome modification of a target sequence in the genome of an Escherichia coli cell. The methods and compositions employ a guide RNA/Cas endonuclease system in combination with a circular polynucleotide modification template to provide an effective system for editing target sites within the genome of an Escherichia coli cell.