Dual-Cut Retron Editors for Large Genomic Insertions and Deletions
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Solution Overview
Problem
Current CRISPR-related mutation/repair methods, particularly those using single-cutters, are inefficient for inserting large DNA fragments and limited in the range of genomic alterations they can achieve, lacking the ability to generate DNA repair templates in situ.
Innovation Solution
The use of dual-cutters, where two guide RNAs target two sites in the genome, with a modified retron non-coding RNA (ncRNA) encoding homology arms and a donor DNA template, allowing for in situ synthesis of donor DNA through reverse transcription, enhancing the efficiency of genomic editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If single-cutter CRISPR methods are used, then the system is simple to operate, but the efficiency of inserting large DNA fragments is substantially reduced
Solution Approach 1:
The patent divides the CRISPR system into multiple cutters (dual-cutters) that target two distinct sites in the genome simultaneously. This segmentation allows for more efficient insertion of large DNA fragments by creating two boundaries for integration, resolving the contradiction between operational simplicity and insertion efficiency.
2Device complexity
If single-cutter methods are used, then the device complexity is low, but the range of genomic alterations is restricted
Solution Approach 1:
The patent implements dual-cutter CRISPR systems that can perform multiple functions: deleting DNA between two cut sites, inserting large fragments at either cut site, or replacing genomic sequences. This multi-functionality expands the range of achievable genomic alterations while maintaining relatively simple system architecture.
3Manufacturing precision
If exogenously delivered DNA repair templates are used, then gene-sized modifications can be achieved, but the ability to generate DNA repair templates in situ is lost
Solution Approach 1:
The patent enables cells to generate their own DNA repair templates in situ through the expression of DNA repair template-encoding sequences within the cell. This self-service approach eliminates the need for exogenous delivery while maintaining the capability to achieve gene-sized modifications, resolving the contradiction between precision and adaptability.
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 significantly improves the efficiency of genomic editing, enabling large insertions and deletions, with up to 200-fold improvement compared to single-cutter methods, and allows for precise genomic alterations.
Implementation Method 1
A portion of the ncRNA can also encode a donor DNA that can be reverse transcribed within the cell to provide the donor DNA
Data Source
AI summary
Described herein are retron-related constructs, expression systems, and methods for precisely deleting, inserting and/or replacing genomic DNA within cells. The inventors have found that CRISPR “single-cutter” methods are substantially less efficient at insertion/replacement of large fragments in comparison to the dual-cutter constructs, expression systems, and methods described herein.