RNA-Guided DNA Transposition for Stable Bacterial Gene Overexpression
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Solution Overview
Problem
Existing genome-scale overexpression (OE) systems in bacteria are not targeted, systematic, or practical, and existing CRISPRa-based OE in bacteria require direct interactions between activator proteins and RNA polymerase, leading to inefficiencies and variations in gene activation.
Innovation Solution
A system utilizing a first non-replicating plasmid expressing an engineered CRISPR-Cas protein and a transposase, and a second non-replicating donor plasmid with a guide RNA and cargo sequence, enabling targeted insertion of promoters or protein expression sequences into bacterial genomes using RNA-guided DNA transposition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPRa-based overexpression is used in bacteria, then gene activation can be achieved, but direct interactions between activator proteins and RNA polymerase are required which lead to stringent spacing and orientation requirements and substantial variation in activity
Solution Approach 1:
The patent extracts the cargo sequence (promoter or protein expression sequence) from the plasmid-based CRISPRa system and delivers it directly to the bacterial chromosome at the target gene locus. This eliminates the need for continuous plasmid maintenance and reduces complexity by removing the requirement for activator proteins that must interact with RNA polymerase. The system uses RNA-guided transposition to place the cargo sequence in cis with the target gene, ensuring stable and reliable overexpression without the variability associated with plasmid copy number fluctuations.
Solution Approach 2:
The patent creates a chromosomal copy of the cargo sequence at the target gene location through RNA-guided transposition. Instead of relying on plasmid-based expression systems that require continuous replication and maintenance, the cargo sequence is copied and integrated into the bacterial chromosome, providing stable inheritance and consistent expression across cell divisions without the need for antibiotic selection.
2Quantity of substance
If plasmid-based overexpression systems are used, then gene overexpression can be achieved, but copy number variation increases experimental noise and antibiotic selection is required for maintenance
Solution Approach 1:
The patent extracts the overexpression function from the plasmid backbone and integrates it directly into the bacterial chromosome at the target gene locus. This eliminates the problems of plasmid copy number variation and antibiotic selection requirements. The cargo sequence (promoter or protein expression sequence) is delivered in cis with the target gene through RNA-guided transposition, ensuring that expression levels are determined by the cargo sequence itself rather than plasmid copy number, thereby improving expression consistency and reducing experimental noise.
3Ease of manufacture
If random transposon integration is used for overexpression, then cargo sequences can be inserted into the genome, but insertions in intergenic regions are required to avoid disrupting adjacent genes which reduces efficiency
Solution Approach 1:
The patent introduces a guide RNA as an intermediary that directs the transposon to the precise location upstream of the target gene. The guide RNA contains a spacer sequence complementary to the target gene's promoter region, enabling specific recognition and binding. This intermediary mechanism allows the transposon to be inserted at the exact desired location (in cis with the target gene) rather than requiring random integration, thereby achieving both high insertion efficiency and precise location control without disrupting adjacent genes.
4Reliability
If engineered transcription factors are used for bacterial CRISPRa, then spacing requirements are partially mitigated and activation is increased, but less than half of targeted endogenous genes could be upregulated and substantial variation in activity remains
Solution Approach 1:
The patent extracts the cargo sequence (promoter or protein expression sequence) and delivers it directly to the target gene locus in the bacterial chromosome through RNA-guided transposition. This eliminates the need for engineered transcription factors and their complex interactions with RNA polymerase. By placing the cargo sequence in cis with the target gene, the system achieves reliable upregulation of targeted genes without the limitations of transcription factor-based approaches, including the inability to upregulate certain genes and substantial activity variation.
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 provides efficient, targeted overexpression of genes in bacteria, improving experimental accuracy and reducing experimental noise, and can deliver cargo sequences such as promoters or protein expression sequences without requiring plasmid replication or leaving exogenous components in the recipient bacteria.
Implementation Method 1
wherein the gRNA is specific for a target site
Data Source
AI summary
Described herein is a system for RNA-guided DNA transposition in a bacterial cell's genome including a) a first non-replicating plasmid expressing an engineered Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) (CRISPR-Cas) protein and a transposase; and b) a second non-replicating donor plasmid, the second non-replicating donor plasmid including an antibiotic selection marker, a guide RNA (gRNA) sequence and a cargo sequence flanked by a right transposon end sequence and a left transposon end sequence, wherein the gRNA is specific for a target site, and wherein the cargo sequence comprises a promoter, a protein expression sequence, or a combination thereof. The first non-replicating plasmid and the second non-replicating donor plasmid, when introduced into a cell, provide insertion of the cargo sequence into the bacterial cell's genome as directed by the gRNA.