Single-Vector Type I CRISPR Delivery for Coordinated Cas Expression
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Solution Overview
Problem
Existing CRISPR/Cas systems often require multiple vectors for efficient delivery and expression of Cas and Cascade proteins, leading to potential interference from host cell mechanisms and reduced efficacy in target cell modification.
Innovation Solution
A single-vector nucleic acid vector is developed, incorporating a Cas3 and Cascade protein-encoding sequences under the control of promoters, allowing coordinated expression via an operon to enhance delivery and efficiency in target cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple vectors are used to deliver CRISPR/Cas components, then expression of Cas and Cascade proteins can be achieved, but delivery complexity increases and host cell interference occurs
Solution Approach 1:
The patent combines multiple CRISPR/Cas components (Cas3 nuclease, Cascade complex, and guide RNA) into a single viral vector system. This merging approach maintains reliable protein expression while simplifying delivery by eliminating the need for multiple separate vectors, thereby reducing host cell interference and improving overall system efficiency
2Reliability
If multiple vectors are used for CRISPR/Cas delivery, then protein expression is achieved, but efficacy in target cell modification is reduced
Solution Approach 1:
By consolidating all essential CRISPR/Cas components into one vector, the patent ensures that all necessary elements are delivered simultaneously to target cells, maintaining high protein expression while maximizing modification efficacy through coordinated delivery and expression of the complete functional system
Solution Approach 2:
The single vector system pre-assembles all CRISPR/Cas components in the delivery vehicle, ensuring that the complete functional complex is introduced to target cells in a coordinated manner, which enhances the efficiency of target sequence modification compared to sequential delivery from multiple vectors
3Productivity
If Cas3 and Cascade proteins are expressed from separate vectors, then protein production is achieved, but expression coordination is poor
Solution Approach 1:
The patent merges the expression cassettes for Cas3 and Cascade proteins into a single vector under coordinated promoter control, ensuring that both proteins are produced in appropriate stoichiometric ratios and temporal patterns, thereby achieving both high productivity and stable expression coordination
Solution Approach 2:
Within the single vector, the patent segments the expression system into distinct but coordinated modules (Cas3 expression cassette, Cascade expression cassette, and guide RNA expression cassette), each under appropriate promoter control, allowing independent optimization while maintaining overall coordination through unified vector delivery
Data Source
AI summary
The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.