Programmable DNA Binding Proteins Enhance Genome Editing
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Solution Overview
Problem
Existing programmable endonucleases, such as CRISPR/Cas systems, face challenges in accessing and efficiently modifying target genomic sequences in human cells due to chromatin structure barriers, leading to reduced activity and increased off-target effects.
Innovation Solution
A composition comprising a programmable DNA modification protein and at least one programmable DNA binding protein, where the DNA binding protein lacks nuclease activity and is targeted to sites proximal to the target chromosomal sequence, enhancing the accessibility of the DNA modification protein to the target site.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If programmable endonucleases (e.g., CRISPR/Cas systems) are used for targeted genome modification, then genome editing capability is improved, but accessibility to target sequences is reduced due to chromatin structure barriers
Solution Approach 1:
The patent introduces chromatin remodeling complexes as intermediary proteins that bind to chromatin regions and modify their structure. These intermediaries facilitate the access of programmable endonucleases to target sequences by altering chromatin accessibility, thereby resolving the barrier between the nuclease and the DNA target without directly enabling the nuclease to penetrate closed chromatin structures.
Solution Approach 2:
The patent employs chromatin remodeling complexes to perform preliminary actions on chromatin structures before the programmable endonucleases arrive at or bind to their target sequences. By pre-modifying chromatin accessibility in the vicinity of target sites, the system prepares the chromatin landscape to be more receptive to subsequent nuclease binding and activity, thus overcoming chromatin-based accessibility barriers.
2Productivity
If programmable endonucleases are used for targeted genome modification, then genome editing capability is improved, but specificity is reduced due to increased off-target effects
Solution Approach 1:
The patent incorporates feedback mechanisms through the use of programmable DNA binding proteins that can monitor and respond to the binding status and activity of programmable endonucleases. These feedback systems allow for real-time adjustment and optimization of nuclease-target interactions, enabling the system to distinguish between true target sites and off-target sequences more effectively, thereby improving specificity while maintaining editing capability.
3Reliability
If chromatin structure barriers are present, then accessibility of programmable endonucleases is reduced, but the complexity of overcoming these barriers increases
Solution Approach 1:
The patent segments the chromatin remodeling function into distinct, modular complexes that can independently bind to and modify specific chromatin regions. By dividing the complex task of chromatin accessibility into separate functional units (e.g., different chromatin remodeling complexes for different chromatin states), the system reduces the overall complexity of interacting with chromatin structures while maintaining effective accessibility to target sequences.
Data Source
AI summary
Compositions and methods for using programmable DNA binding proteins to increase the efficiency and/or specificity of targeted genome modification or to facilitate the detection of specific genomic loci in eukaryotic cells.