Mobile CRISPRi Plasmids for Stable Bacterial Integration
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Solution Overview
Problem
Current CRISPRi systems face challenges in establishing effective and stable genomic integration across diverse bacteria, limiting their widespread use for dissecting bacterial gene function and studying microbiome interactions.
Innovation Solution
Development of CRISPRi systems and methods that enable modular, stable genomic integration and ease of transfer to diverse bacteria through conjugation, using artificial DNA constructs with sgRNAs, dCas9, antibiotic resistance genes, and nucleotide transfer sequences like Tn7L and Tn7R transposon sequences or ICE elements, facilitating genetic dissection and analysis of bacterial physiology and host-microbe interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPRi systems are established in bacteria, then gene expression blocking capability is achieved, but stable genomic integration and ease of transfer to diverse bacteria remain difficult
Solution Approach 1:
The patent creates a universal CRISPRi system that functions across diverse bacterial species by using a standardized plasmid backbone (pJMP1337) with broad-host-range conjugation elements. The system maintains consistent sgRNA-dCas9 repression mechanism while adapting to different bacterial hosts through mobile genetic elements, enabling one system to serve multiple bacterial species.
Solution Approach 2:
The patent introduces mobile genetic elements (Tn7 transposon, ICE Bs1) as intermediaries that mediate the transfer of CRISPRi components between bacteria. These elements act as bridges, carrying the CRISPRi plasmid from donor to recipient cells through conjugation, thereby enabling cross-species transfer without requiring species-specific adaptation of the core repression mechanism.
2Ease of operation
If CRISPRi components are transferred between bacteria, then ease of transfer is improved, but stable genomic integration is compromised
Solution Approach 1:
The patent incorporates mobile genetic elements (Tn7 transposon, ICE Bs1) into the plasmid design beforehand, preparing the CRISPRi construct for efficient conjugative transfer. These pre-integrated elements enable the plasmid to self-transfer between bacteria without requiring additional manipulation, while the plasmid maintains its ability to stably maintain CRISPRi components during and after transfer.
3Adaptability or versatility
If mobile genetic elements are used for transfer, then ease of transfer to diverse bacteria is improved, but plasmid size increases
Solution Approach 1:
The patent segments the CRISPRi system into modular components: the core repression elements (sgRNA, dCas9), the mobile genetic elements (Tn7 transposon or ICE Bs1), and the plasmid backbone. This segmentation allows the mobile elements to be optimized for transfer efficiency while the core CRISPRi components remain compact, enabling the system to achieve broad transferability without excessive plasmid size increase.
Data Source
AI summary
Described in this disclosure are CRISPRi systems and methods, along with the related compositions and kits, that combine modularity, stable genomic integration, and ease of transfer to diverse bacteria by conjugation. CRISPRi compositions, methods, systems and kits described herein allow for genetic dissection of bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, as well as comprehensive screening for host-microbe interactions. Embodiments of the invention comprise compositions, methods, systems, and kits for CRISPRi-based repression of gene expression in bacteria.