Triple Cross DNA Construct for Scarless Microorganism Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genetic tools for manipulating the genomes of non-model microorganisms, such as Clostridium bacteria, are rudimentary and inefficient, requiring multiple steps and often resulting in cumbersome mutant screening processes and fickle transformation steps, limiting their application in medical, chemical, and industrial contexts.
Innovation Solution
A method utilizing homologous recombination with a DNA construct containing three homology arms and two counter selection markers to efficiently insert, replace, or delete nucleic acid sequences in microorganisms, allowing for 'scarless' modification and nearly 100% efficiency without leaving residual artifacts or requiring genome preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing genetic tools (ClosTron, ACE, counter selection markers) are used for Clostridium manipulation, then basic genetic modification is achievable, but the process requires multiple steps, cumbersome screening, and shows low reliability
Solution Approach 1:
The patent combines multiple genetic manipulation functions (deletion, insertion, replacement) into a single integrated DNA construct system with three homology arms and dual counter-selection markers, eliminating the need for separate ClosTron and ACE steps and achieving reliable single-step modification
Solution Approach 2:
The DNA construct design with three homology arms (LHA, RHA1, RHA2) and dual counter-selection markers (CS1, CS2) provides universal applicability for various genetic manipulation types (deletion, insertion, replacement) across different Clostridium species, replacing multiple specialized tools with one versatile system
2Productivity
If existing genetic tools are used, then rudimentary modification is possible, but transformation efficiency is low and mutant screening is time-consuming
Solution Approach 1:
The dual counter-selection marker system provides immediate feedback through selectable phenotypes: CS1 (sacB) eliminates transformants without proper integration, while CS2 (ccdB) eliminates those with incomplete modification, enabling rapid identification of successful mutants without time-consuming screening
Solution Approach 2:
The patent optimizes homology arm lengths (LHA: 50-2000 bp, RHA1: 50-2000 bp, RHA2: 50-2000 bp) and uses specific counter-selection markers with well-characterized selection parameters to maximize transformation efficiency and minimize screening time across different Clostridium species
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 robust and efficient genetic manipulation of microorganisms, allowing for precise modification at any site with minimal undesired integrations, reducing the complexity and time required for genetic modifications, and achieving high recombination efficiency.
Implementation Method 1
allowing the genetic element of (a) to undergo homologous recombination with the DNA construct of (b), whereby T1 aligns with LHA1 and T2 aligns with RHA1 to insert the portion of the DNA construct between LHA1 and RHA1
Implementation Method 2
allowing the genetic element of (c) to undergo self-homologous recombination, whereby T3 aligns with RHA2 to remove the portion of the genetic element between T3 and RHA2
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The invention provides a genetic tool to insert, replace, delete, or otherwise manipulate a nucleic acid sequence in a microorganism to produce a recombinant microorganism. Notably, the invention makes use of homologous recombination, a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Since the invention involves three homologous recombination events, it is referred to as a "triple cross" method.