Inducible CRISPR/Cas System for C1-Fixing Bacteria
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Solution Overview
Problem
Existing CRISPR/Cas systems are toxic to C1-fixing bacteria when using constitutive promoters, hindering genetic modification of these microorganisms.
Innovation Solution
Development of a CRISPR/Cas system using an inducible promoter, such as a tetracycline inducible promoter, to control the expression of the Cas9 protein, allowing for targeted genetic engineering of C1-fixing bacteria like Clostridium autoethanogenum, enabling precise genome editing through homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a constitutive promoter is used to drive Cas9 expression in CRISPR/Cas system, then the genetic modification capability is achieved, but the C1-fixing bacteria experience toxicity and reduced viability
Solution Approach 1:
The patent applies dynamics by replacing the constitutive promoter with an inducible promoter (such as Ptac or Ptet) that allows dynamic control of Cas9 expression. The system transitions from static continuous expression to dynamic regulated expression, enabling the bacteria to maintain viability while achieving genome editing capability when induction is applied.
Solution Approach 2:
The patent changes the expression parameter of Cas9 from constant high-level expression (constitutive promoter) to conditionally regulated expression (inducible promoter). By controlling induction conditions (such as IPTG concentration or tetracycline presence), the system optimizes the balance between editing efficiency and bacterial toxicity, effectively resolving the contradiction.
2Adaptability or versatility
If CRISPR/Cas system components are introduced into C1-fixing bacteria, then genetic engineering capability is enabled, but the system complexity increases due to multiple vector components
Solution Approach 1:
The patent merges multiple CRISPR/Cas system components (Cas9 gene, inducible promoter, guide RNA expression cassette, and homology arms) into integrated vector systems. This consolidation reduces the number of separate transformation steps and simplifies the overall system while maintaining full genetic engineering functionality.
Solution Approach 2:
The patent designs universal vector systems that can be applied across different C1-fixing bacteria species (such as Clostridium autoethanogenum, C. ljungdahlii, and C. ragsdalei). The modular vector design with standardized components enables broad adaptability while reducing system complexity through reuse of proven elements.
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 inducible CRISPR/Cas system effectively enables gene deletion, insertion, and expression in C1-fixing bacteria, improving the efficiency of genetic modification and reducing toxicity, with demonstrated efficiencies of up to 60% in specific examples.
Implementation Method 1
a nucleotide sequence encoding a guide RNA that hybridizes with the target sequence
Implementation Method 2
catalytically active Cas9... may be used to cleave the DNA molecule
Implementation Method 3
the 5′ homology arm and the 3′ homology arm hybridize with the DNA molecule and homologous recombination occurs, resulting in the replacement of the target sequence
Data Source
AI summary
The invention provides methods of genetically engineering a C1-fixing bacterium using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) system. Preferably, the Cas protein is under the control of an inducible promoter.