Repair-Modulating Enzyme Molecules Enhance Genome Editing Precision
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Solution Overview
Problem
Current CRISPR/Cas systems for genome editing in eukaryotic cells face inefficiencies in modifying nucleic acids, limiting their broader application beyond research use.
Innovation Solution
A genome editing system comprising a RNA-guided nuclease molecule, a guide RNA molecule, and a heterologous Repair-Modulating Enzyme Molecule (RMEM) is used to modulate DNA repair pathways, allowing for precise control over nucleic acid alterations by contacting cells with the RMEM to alter or repair nucleic acids at target positions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas system is used for genome editing in eukaryotic cells, then site-specific double strand breaks can be introduced to enable nucleic acid alteration, but the efficiency of genome modification remains insufficient for broader application
Solution Approach 1:
The patent introduces repair-modulating enzyme molecules as intermediary components that mediate between the Cas9-induced double strand break and the final repair outcome. These enzyme molecules (such as TdT, Artemis, or other DNA modification enzymes) are delivered to cells along with Cas9 and guide RNA, where they modulate the DNA repair pathways to achieve desired editing outcomes with higher efficiency and precision.
Solution Approach 2:
The patent creates a composite genome editing system that combines multiple functional components: Cas9 nuclease, guide RNA, and repair-modulating enzyme molecules. This composite approach allows the system to simultaneously perform DNA cleavage and modulate repair pathways, achieving both high efficiency and reliability in genome modification that neither component could achieve alone.
2Manufacturing precision
If DNA repair pathways are allowed to operate naturally after Cas9 cleavage, then cells can repair the breaks through NHEJ or HDR, but precision and control over the editing outcomes are limited
Solution Approach 1:
The patent alters the parameters of DNA repair by introducing enzyme molecules that change the biochemical conditions and mechanisms of repair. For example, TdT enzyme adds nucleotides to DNA ends, Artemis processes DNA ends differently, and other enzymes modify repair kinetics and pathways. These parameter changes enable precise control over whether deletions, insertions, or specific sequence corrections occur at the editing site.
Solution Approach 2:
The system incorporates feedback control by using repair-modulating enzymes that respond to the specific context of the DNA break and the desired editing outcome. The enzymes are designed to modulate repair pathways based on the local DNA structure and sequence context, providing feedback-based control that enhances precision while maintaining ease of operation through a single delivery system.
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 enhances the efficiency of genome editing by modulating DNA repair pathways, allowing for increased precision in altering nucleic acids, including suppressing or enhancing deletions, insertions, gene conversion, and gene correction at specific target positions.
Implementation Method 1
an RNA-guided nuclease molecule (e.g., a Cas9 molecule) may be used to mediate a break near a position that one desires to edit
Implementation Method 2
The cell then repairs the break through one of several DNA repair pathways, such as non-homologous end-joining (NHEJ) or homology-directed repair (HDR)
Implementation Method 3
The cell then repairs the break through one of several DNA repair pathways, such as non-homologous end-joining (NHEJ) or homology-directed repair (HDR)
Implementation Method 4
Provided herein is a genome editing system comprising a RNA-guided nuclease (e.g., a Cas9 molecule), at least one gRNA molecule, and a repair-modulating enzyme molecule (RMEM). It is believed that by contacting a cell, or a population of cells, with a RMEM, the ability of certain cellular DNA repair pathways to resolve or repair a RNA-guided nuclease-mediated cleavage event can be modulated.
Data Source
AI summary
This application provides improved methods of genome editing. The genome editing systems described herein comprise a RNA-guided nuclease molecule and a Repair-Modulating Enzyme Molecule (RMEM).