E. Coli Gene Editing with Cas12k-ShCAST for Large DNA Integration

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Solution Overview

Problem

Existing gene editing systems for Escherichia coli, such as CRISPR/Cas9 and ShCAST, face challenges in achieving efficient and precise integration of large DNA cargo, particularly in strains like BL21(DE3) and W, due to DNA repair abilities and poor integration at specific chromosomal positions.

Innovation Solution

A gene editing system comprising a helper plasmid with enhanced Cas12k expression and a donor plasmid, utilizing independent promoters and optimized editing temperatures, to facilitate precise integration of exogenous genes into Escherichia coli strains, including BL21(DE3), by forming a ShCAST transposon protease complex and targeting specific chromosomal sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If CRISPR/Cas9 technology is used to integrate large DNA cargo into Escherichia coli, then the cargo size can be increased to 10kb, but CRISPR-induced double-strand break may trigger genome instability and integration efficiency is relatively low in strains such as BL21(DE3) and W

Engineering Contradiction:

Improvecargo sizeVSAvoidgenome stability

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent changes the molecular mechanism parameter from CRISPR/Cas9-induced double-strand break to transposase-mediated transposition, which integrates DNA without causing genome instability. This parameter change allows maintaining large cargo size while improving genome stability and integration efficiency in difficult-to-transform strains.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If ShCAST technology is used for on-target DNA transposition without inducing DSB, then genome instability is avoided, but integration at specific chromosomal positions is poor

Engineering Contradiction:

Improvegenome stabilityVSAvoidintegration precision

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent merges the advantages of two systems: transposase-mediated transposition (for genome stability) and CRISPR/Cas9 (for precise targeting). The combined system uses Cas12k protein with sgRNA for specific chromosomal position recognition, while TnsB-TnsC-TniQ complex performs the actual integration without inducing DSB, achieving both high integration precision and genome stability.

Inventive Principle:

Principle #5Merging (Combining)

3Quantity of substance

If CRISPR/Cas9/λ-Red system is leveraged for genetic engineering, then the size of integrated DNA is increased, but integration efficiency is relatively low in strains with varying DNA repair abilities

Engineering Contradiction:

Improveintegrated DNA sizeVSAvoidintegration efficiency

Core Design Contradiction:

Quantity of substanceVSProductivity

Solution Approach 1:

The patent replaces the λ-Red recombineering mechanism with transposase-mediated transposition. This substitution eliminates the dependency on host DNA repair pathways, enabling high integration efficiency across different E. coli strains regardless of their varying DNA repair abilities, while maintaining the capability to integrate large DNA fragments.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

The system achieves high on-target efficiency and stable integration of DNA up to 14.5 kb, enhances metabolic engineering by knocking out genes contributing to byproduct production, and improves recombinant protein production in strains like BL21(DE3) and MG1655, while minimizing off-target effects.

Implementation Method 1

A sequence of the spacer is homologous to a first specific sequence of a chromosome of the Escherichia coli

Methodology Applied

Scientific EffectSequence complementarity:

Implementation Method 2

the helper plasmid expresses a TnsB protein, a TnsC protein, a TniQ protein and a Cas12k protein to form a ShCAST transposon protease complex, the helper plasmid expresses the sgRNA, the donor plasmid expresses the sgRNA, and the exogenous gene is inserted into a first specific sequence of the transformant

Methodology Applied

Scientific EffectTransposition:

Data Source

PatentUS20260035708A1Gene Editing System of Escherichia Coli and Gene Editing Method Thereof

Publication Date: 2026.02.05 NATIONAL TSING HUA UNIVERSITY

AI summary

A gene editing system of Escherichia coli includes an Escherichia coli, a helper plasmid and a donor plasmid. The helper plasmid successively includes a transposase complex expression cassette, a Cas12k expression cassette, a first sgRNA cassette, a first antibiotic resistance gene and a first replication origin. The donor plasmid successively includes a left end sequence of a ShCAST transposon, an exogenous gene expression cassette, a right end sequence of the ShCAST transposon, a second sgRNA cassette, a second antibiotic resistance gene and a second replication origin.