Modulating Cellular Repair for Genomic Editing Efficiency
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid-guided nuclease editing methods face low efficiency due to native cellular repair mechanisms, such as DNA mismatch repair (MMR) systems, which impede editing and produce undesirable byproducts, resulting in low rates of precise edits in cell populations.
Innovation Solution
The use of a system comprising a CREATE fusion gRNA (CFgRNA), a nickase-RT fusion enzyme, and a mismatch repair (MMR) perturbation agent, including wild-type or variant MMR polypeptides, to disrupt cellular MMR pathways, thereby enhancing editing efficiency by perturbing native repair mechanisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nuclease editing is performed using standard methods, then genomic editing can be achieved, but the percentage of precisely edited cells remains low due to native cellular repair mechanisms
Solution Approach 1:
The patent extracts and removes the harmful MMR repair mechanism from the cellular environment by introducing MMR-inhibiting peptides or small molecules. This extraction of the detrimental repair pathway allows the editing system to function without interference, thereby increasing both the precision and percentage of successfully edited cells
Solution Approach 2:
The patent introduces intermediary substances (MMR-inhibiting peptides or small molecules) that mediate between the editing system and the cellular repair machinery. These intermediaries selectively inhibit MMR activity at the editing site without completely disabling the cell's repair functions, thus improving editing efficiency while maintaining cellular health
2Reliability
If native cellular MMR systems are active during editing, then cellular repair functions are maintained, but editing efficiency is impeded and undesirable byproducts are produced
Solution Approach 1:
The patent applies partial inhibition of MMR systems rather than complete suppression. By using MMR-inhibiting peptides or small molecules at controlled concentrations, the system achieves sufficient inhibition to improve editing efficiency while leaving residual repair functions intact to maintain cellular reliability and prevent accumulation of harmful mutations
Solution Approach 2:
The patent employs preliminary action by pre-treating cells with MMR-inhibiting agents before introducing the editing system. This preliminary inhibition of MMR pathways prepares the cellular environment to be more receptive to editing, reducing interference during the editing process and improving overall efficiency
3Stability of the object's composition
If MMR systems are active during editing, then genomic stability is maintained, but reversion to original sequence and indel formation increase
Solution Approach 1:
The patent converts the harmful effect of MMR systems (which cause reversion and indels during editing) into a benefit by selectively inhibiting MMR activity only during the editing window. After editing is complete, MMR function is restored to maintain genomic stability, thus transforming the previously harmful repair mechanism into a controlled process that benefits editing outcomes
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 increases the percentage of precisely edited cells by overcoming the limitations of MMR systems, achieving improved editing outcomes with higher efficiency and reduced reversion to the original sequence or unwanted indel formation.
Implementation Method 1
a nickase-RT fusion enzyme, the MMR perturbation agent comprising a first MMR polypeptide or a polynucleotide sequence encoding the first MMR polypeptide
Implementation Method 2
a CREATE fusion gRNA (CFgRNA) or a polynucleotide sequence encoding a CFgRNA, the CFgRNA comprising an edit to a target genomic locus
Data Source
AI summary
The present disclosure relates to methods and compositions of matter to increase the percentage of edited cells in a cell population when employing nucleic-acid guided editing methods, as well as systems and instruments for performing these methods and using these compositions.