Recursive Nucleic Acid Editing Vector Curing
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Solution Overview
Problem
There is a need for improved methods and instruments to cure editing vectors in recursive nucleic acid-guided nuclease editing protocols, as existing techniques lack efficiency in eliminating prior editing vectors to allow for subsequent editing without competition.
Innovation Solution
The method involves designing and synthesizing sets of editing cassettes with inducible promoters, assembling them into vector backbones with selectable markers and curing target sequences, and using a nuclease under the control of an inducible promoter to induce transcription and cure the editing vectors, allowing for sequential rounds of editing without interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If editing vectors are not cured between rounds of editing, then the prior editing vectors remain in cells and compete with subsequent editing vectors, but curing processes add complexity to the recursive editing protocol
Solution Approach 1:
The curing gRNA and nuclease are pre-assembled in the engine vector before the editing round begins. The curing mechanism is prepared in advance but only activated after the editing vector has been delivered and editing has occurred, eliminating the need for separate curing steps between rounds.
Solution Approach 2:
The curing components (curing gRNA and nuclease) are merged into the same engine vector that delivers the editing components. This consolidation allows the curing mechanism to be co-delivered with the editing machinery and activated at the appropriate time, simplifying the overall protocol.
2Ease of operation
If curing gRNA and nuclease are pre-assembled in the engine vector, then curing components are readily available for activation, but the engine vector becomes more complex
Solution Approach 1:
The engine vector uses inducible promoters that allow dynamic control of gene expression. The curing gRNA and nuclease are transcribed only when specifically induced, allowing the vector to transition between different functional states (editing-ready vs. curing-active) without permanent structural changes.
Solution Approach 2:
The engine vector is organized into distinct functional modules: editing components (editing gRNA, donor DNA), curing components (curing gRNA, nuclease), and regulatory elements (inducible promoters). This modular segmentation allows each component to be independently optimized and controlled.
3Measurement precision
If inducible promoters are used to control editing and curing, then temporal precision is improved, but the system requires additional regulatory components
Solution Approach 1:
The inducible promoter system serves multiple functions: it controls both editing component expression and curing component expression, and can be induced at different times to achieve different outcomes. This multi-functionality reduces the need for separate regulatory mechanisms for each process.
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 enables effective curing of editing vectors, allowing for precise and targeted genome editing by eliminating prior editing vectors, thereby enabling multiple rounds of editing without competition and improving editing efficiency and vector propagation.
Implementation Method 1
an engine vector and the engine vector comprises a curing gRNA under the control of a second inducible promoter, a nuclease under the control of the third inducible promoter
Implementation Method 2
inducing the third and second inducible promoters thereby inducing transcription of the nuclease and curing gRNA
Implementation Method 3
making cells of choice electrocompetent
Data Source
AI summary
The present disclosure provides automated multi-module instrumentation and automated methods for performing recursive editing of live cells with curing of editing vectors from prior rounds of editing.