CRISPR/Cas Microbial Phenotype Control via Segmented Gene Modules
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Solution Overview
Problem
Current genomic editing technologies, such as CRISPR/Cas, face limitations in precisely and efficiently altering gene expression in microbial organisms, particularly in controlling phenotype changes and repair processes, which are crucial for targeted modifications and functional restoration.
Innovation Solution
Engineered microbial organisms equipped with specific heterologous polynucleotide sequences, including phenotype coding sequences linked to promoters, Cas nuclease, and guide RNA, enable targeted gene alterations and repairs by inducing detectable phenotypes such as color, fluorescence, scent, or antibiotic resistance, using the CRISPR/Cas system in conjunction with lambda red recombineering for precise modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas system is used to induce double-stranded breaks for gene modification, then gene editing capability is improved, but precision and efficiency in controlling phenotype changes deteriorates
Solution Approach 1:
The system segments the gene editing process into distinct functional modules: Cas nuclease for DNA cleavage, guide RNA for target recognition, and separate phenotype coding sequences for controlled expression. This segmentation allows independent optimization of each component, improving both editing capability and phenotypic control precision.
Solution Approach 2:
The patent incorporates phenotype coding sequences and regulatory elements into the editing construct before delivering it to the cell. This preliminary arrangement ensures that once editing occurs, the phenotypic changes are pre-programmed and controlled, rather than relying on random mutations alone.
2Reliability
If multiple phenotype coding sequences are introduced for targeted modifications, then functional restoration capability is improved, but system complexity increases
Solution Approach 1:
The system uses universal regulatory elements (promoters, terminators) that can control multiple different phenotype coding sequences. This multi-functionality allows a single regulatory framework to manage diverse genetic modifications, improving functional restoration while avoiding proportional increases in system complexity.
Solution Approach 2:
Multiple phenotype coding sequences are nested within a single polynucleotide construct or plasmid, along with their regulatory elements. This nesting approach consolidates multiple functions into a unified structure, reducing the overall complexity compared to separate constructs for each phenotype.
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 allows for controlled and efficient alteration of gene expression, enabling the disruption or restoration of specific phenotypes, thereby facilitating precise genetic modifications and visualizing changes in microbial organisms, enhancing the precision and effectiveness of gene editing processes.
Implementation Method 1
The Cas nuclease can specifically induce a DSB at a target genomic locus through the use of a guide nucleic acid that targets the genomic locus
Implementation Method 2
each of the first and the second detectable phenotypes is a detectable color, fluorescence, scent, enzymatic activity, antibiotic resistance, morphology or lethality
Data Source
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AI summary
Engineered microbial organisms for altering gene expression are provided. In some embodiments, the engineered microbial organism comprises: one or more heterologous polynucleotide sequence comprising a phenotype coding sequence operably linked to a promoter; a heterologous polynucleotide sequence comprising an inducible promoter operably linked to a polynucleotide encoding a Cas nuclease; and a heterologous polynucleotide sequence comprising a guide RNA (gRNA) that targets the phenotype coding sequence.