Engineered Nuclease Fusion Proteins for Precise Eukaryotic Editing
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Solution Overview
Problem
Current nucleases used for nucleic acid editing, such as meganucleases, zinc finger nucleases, and CRISPR systems, are limited in efficiency and precision, particularly in eukaryotic cells, necessitating the development of more effective and precise nucleases for genetic engineering and therapeutic applications.
Innovation Solution
Engineered nucleases with specific amino acid sequences, including those with at least 75-90% identity to SEQ ID NOs: 1-304, and fusion proteins with effector domains, such as transcription activators or deaminases, are developed to enhance editing precision and efficiency, accompanied by guide RNAs and localization sequences for targeted nucleic acid modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If current nucleases (meganucleases, ZFNs, TALENs, CRISPR systems) are used for nucleic acid editing, then nucleic acid modification can be achieved, but editing efficiency and precision are limited particularly in eukaryotic cells
Solution Approach 1:
The patent applies parameter changes by systematically optimizing amino acid sequences of engineered nucleases to improve both editing precision and efficiency. Specific amino acid substitutions and sequence variations are introduced to enhance nuclease activity and target specificity in eukaryotic cells, directly addressing the limitation of current nucleases
Solution Approach 2:
The patent employs composite materials by creating fusion proteins that combine engineered nuclease domains with effector domains (such as transcription activators, deaminases, or other functional domains). This composite structure allows the nuclease to perform both targeted DNA binding and additional functions simultaneously, improving overall editing efficiency and precision
2Manufacturing precision
If nucleases are engineered with specific amino acid sequences to improve precision, then editing specificity increases, but device complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the nuclease into modular domains: a DNA-binding domain (such as CRISPR-Cas9 or other nuclease domains) and separate effector domains. This modular architecture allows for precise targeting through the DNA-binding domain while attaching functional effector domains, improving editing specificity without requiring complete redesign of the entire nuclease structure
Solution Approach 2:
The patent implements universality by designing engineered nucleases with universal DNA-binding capabilities that can be paired with various effector domains. The core nuclease structure remains consistent while different effector domains can be attached to achieve different functions (editing, activation, repression), reducing the need to develop entirely new complex structures for each application
Data Source
AI summary
The present disclosure provides components, compositions, methods, and systems thereof for nucleic acid editing. Particularly, the disclosure provides engineered nucleases, fusion proteins of the engineered nucleases, systems including the engineered nucleases, and methods of using thereof.