Iterative Nucleic Acid-Guided Nuclease Editing with Self-Curing Vectors
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Solution Overview
Problem
Existing CRISPR-based nucleic acid-guided cell editing methods are limited to a small number of iterative editing operations and lack mechanisms for reliably introducing new edits over multiple rounds, typically targeting only a few pre-defined genomic loci.
Innovation Solution
A method involving iterative nucleic acid-guided nuclease editing that includes providing cells with editing vectors containing selectable markers and curing gRNAs, allowing for high-efficiency cleavage and removal of previous editing vectors, enabling multiple rounds of genomic edits with high curing efficiency (>99%) and improved editing efficiency (>70%) through the use of nucleic acid-guided nucleases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-based nucleic acid-guided cell editing methods are used, then targeted genomic edits can be introduced, but the methods are limited to a small number of iterative editing operations and lack mechanisms for reliably introducing new edits over multiple rounds
Solution Approach 1:
The editing system is divided into separate functional modules: a nuclease component (e.g., Cas9) and multiple interchangeable editing vectors, each containing a specific editing cassette and a curing gRNA. This segmentation allows the nuclease to be reused across multiple editing rounds while each vector is designed to be temporarily present and then removed, enabling reliable iterative editing.
Solution Approach 2:
Each editing vector contains a curing gRNA that targets and eliminates the vector itself after the editing function is performed. This self-destruct mechanism ensures that previous editing vectors are removed from the cell population, preventing interference with subsequent editing rounds and enabling reliable introduction of new edits.
2Adaptability or versatility
If multiple editing vectors are used for iterative editing, then more genomic loci can be targeted, but previous editing vectors interfere with new editing vectors
Solution Approach 1:
Each editing vector is pre-equipped with a curing gRNA that is designed to target and eliminate that specific vector. This preliminary inclusion of the curing mechanism ensures that when new editing vectors are introduced, the previous vectors are automatically removed, eliminating interference before it can affect the new editing operations.
3Duration of action of stationary object
If editing vectors are maintained across multiple rounds, then editing function is preserved, but curing efficiency decreases and editing reliability is reduced
Solution Approach 1:
The editing process follows a periodic cycle: an editing vector is introduced to perform its editing function, then the curing gRNA eliminates the vector in a periodic removal event. This periodic action pattern allows the vector to persist long enough to fulfill its editing purpose while ensuring it is subsequently removed to maintain high curing efficiency and reliability for the next editing round.
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
Enables reliable and rapid genomic editing over multiple rounds with high curing and editing efficiencies, facilitating rapid diversification and engineering of cell genomes, exceeding traditional combinatorial protein engineering methodologies.
Implementation Method 1
curing the first editing vector comprises cleaving the first editing vector at the first selectable marker with a nucleic acid-guided nuclease guided by the first curing gRNA
Data Source
AI summary
The present disclosure provides systems, methods, and compositions for performing iterative genomic editing of live cells with curing of editing vectors from prior rounds of editing.