Re-Editable Template Sequences for Precise Multi-Round Genome Editing
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Solution Overview
Problem
Existing genome editing technologies face challenges such as unwanted cleavage at previously edited sites and off-target effects, as well as limitations in programmable epigenome editing requiring constitutive expression, which complicates therapeutic applications.
Innovation Solution
A flexible genome editing technology using a re-editable template sequence with no substantial sequence similarity to the genome, allowing precise and specific editing, and enabling subsequent modifications by different genome editing systems, minimizing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If genome editing technologies (ZFN, TALEN, CRISPR) are used to achieve precise DNA binding and editing, then specific DNA binding and precise genome targeting are improved, but unwanted cleavage at previously edited sites and off-target effects occur
Solution Approach 1:
The patent applies preliminary action by inserting a re-editable template sequence into the target locus before performing the actual genome editing. This template sequence serves as a pre-prepared structure that guides subsequent editing operations, allowing the same or different nucleases to be used multiple times without causing off-target effects at previously edited sites
Solution Approach 2:
The re-editable template sequence acts as an intermediary element between the nuclease and the target locus. This template sequence with unique sequence and flanking homology arms mediates the interaction, enabling precise targeting while preventing unwanted cleavage by providing a structured recognition site that differs from the original genomic sequence
2Manufacturing precision
If sequence-specific recombinases and integrases are used for site-specific integration, then specific genomic sequence targeting is improved, but residual sequences from integration remain (non-scarless)
Solution Approach 1:
The patent applies local quality by designing the re-editable template sequence with specific local characteristics - a unique sequence portion for nuclease recognition and flanking homology arms for integration. This local differentiation allows precise targeting at the specific locus while enabling clean integration without leaving residual sequences from the integration event
3Object-affected harmful factors
If programmable epigenome editing is used to avoid DNA breaks, then cellular toxicity is reduced, but constitutive expression of dCas-fusion proteins is required
Solution Approach 1:
The patent substitutes the mechanical system of constitutive protein expression with a genetic template-based approach. Instead of requiring continuous expression of dCas-fusion proteins, the re-editable template sequence embedded in the genome provides a stable, heritable record of the editing target, allowing transient or inducible nuclease expression to achieve the same effect without continuous protein production
4Adaptability or versatility
If multiple different types of mutations are developed in platform cells, then versatility for treating multiple indications is improved, but manufacturing time and costs increase
Solution Approach 1:
The patent applies universality by creating a re-editable template sequence that can serve multiple functions - it enables the same platform cell to be edited by different nucleases (ZFN, TALEN, CRISPR) to generate various mutations for different indications. The template sequence with its unique region and flanking homology arms provides a universal interface for diverse editing tools, reducing the need for separate cell lines for each indication
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 rapid development of multiple mutations in platform cells for therapeutic use, reducing manufacturing time and costs, and providing a universal off-the-shelf therapeutic solution.
Implementation Method 1
Cas and related nucleases are targeted to specific sites by an RNA guide molecule that base-pairs directly with the target locus
Implementation Method 2
methods of engineering a re-editable cell comprising a unique re-editable template sequence flanked by two homology arms such that the re-editable template sequence is inserted into the target locus by homologous recombination
Data Source
AI summary
The present invention provides, among other things, methods of engineering a re-editable target locus in a cell, comprising inserting an exogenous re-editable template sequence to a target locus and methods of engineering a re-editable cell comprising a re-editable template sequence flanked by two homology arms such that the re-editable template sequence is inserted into a target locus by homologous recombination, and methods of using the same. In some aspects, provided is a re-editable template sequence, wherein the template sequence has no substantial sequence similarity to any region in the genome, and wherein the template sequence is recognizable by a genome editing system.