Curing Vector for Nucleic Acid Editing in E. coli
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Solution Overview
Problem
Current methods for nucleic acid-guided nuclease gene editing lack efficient ways to cure prior editing components and vectors, making it difficult to return cells to their native state after editing and to prepare them for subsequent rounds of editing.
Innovation Solution
The development of compositions and methods involving curing vectors and automated multi-module instrumentation that allow for the precise removal of editing and engine vectors, using anti-target gRNA, temperature-sensitive origins of replication, and distinct antibiotic resistance genes to restore cells to their native state and prepare them for recursive editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nuclease editing is performed to make precise genomic changes, then gene function manipulation is achieved, but editing components and vectors remain in the cells preventing subsequent editing rounds
Solution Approach 1:
The patent introduces a curing gRNA and curing nuclease system that is pre-designed to target and eliminate editing components before subsequent editing rounds. The curing plasmid is prepared in advance with specific gRNA sequences that recognize and bind to editing vector sequences, enabling systematic removal of prior editing components through controlled expression of the curing nuclease.
Solution Approach 2:
The patent uses a curing plasmid as an intermediary carrier that delivers the curing gRNA and curing nuclease components into the cells. This intermediary system facilitates the removal of editing vectors without directly interfering with the genomic editing process, acting as a temporary vehicle that can be introduced, executed its curing function, and then eliminated.
2Productivity
If editing vectors are retained in cells for multiple rounds of editing, then recursive editing capability is maintained, but cells cannot be returned to native state and antibiotic resistance genes persist
Solution Approach 1:
The patent employs a curing mechanism that specifically extracts and removes editing vectors and antibiotic resistance genes from cells between editing rounds. The curing gRNA targets sequences unique to the editing vectors, directing the curing nuclease to cleave and eliminate these foreign elements while preserving the intended genomic edits and native cellular components.
Solution Approach 2:
The patent implements a system where editing vectors are deliberately discarded after serving their editing purpose, and cells are recovered in their native state for subsequent editing rounds. The curing process systematically eliminates vector backbone sequences, antibiotic resistance markers, and other foreign DNA elements, allowing cells to be reused without carrying over harmful or interfering components.
3Ease of operation
If curing plasmids are used to eliminate editing vectors, then editing components are removed, but the curing plasmid itself must be cured to return cells to completely native state
Solution Approach 1:
The patent incorporates a temperature-sensitive origin of replication in the curing plasmid that is designed to become non-functional at elevated temperatures. This preliminary design feature allows the curing plasmid to be easily eliminated by simply shifting the growth temperature, avoiding the need for additional curing agents or complex removal protocols.
Solution Approach 2:
The patent utilizes a temperature-sensitive origin of replication that undergoes a phase transition in functionality based on temperature. At permissive temperatures (e.g., 37°C), the origin supports plasmid replication; at restrictive temperatures (e.g., 42°C), the origin becomes non-functional, causing automatic plasmid loss as cells fail to replicate the curing plasmid during division.
Data Source
AI summary
The present disclosure provides compositions of matter, methods, modules and automated multi-module instrumentation for performing editing of live cells followed by curing of editing and engine vectors from prior rounds of editing, followed by curing of the curing vector.