Engineered Nuclease Compositions for Precise Eukaryotic Editing
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Solution Overview
Problem
CRISPR/Cas systems face limitations in eukaryotic organisms, including inefficient delivery to mature cells, low editing efficiency, off-target events, and target sequence preferences, as well as suboptimal enzymatic activity conditions.
Innovation Solution
Engineered nucleases with amino acid sequences having at least 70-99% identity to specific SEQ IDs, and specific substitutions at key positions, enhancing their efficiency and specificity for nucleic acid modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas systems are used for nucleic acid editing, then versatility and ease of use are improved, but delivery efficiency to mature cells and editing efficiency are worsened
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the Cas9 nuclease through specific substitutions (e.g., positions 164, 271, 559, 564, 570, 827) to optimize enzymatic activity and cellular delivery. These sequence variations create engineered nucleases with improved properties while maintaining the core CRISPR/Cas functionality, thereby resolving the contradiction between versatility and editing efficiency.
2Ease of operation
If CRISPR/Cas systems are used for nucleic acid editing, then ease of operation is improved, but off-target events increase
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at key positions within the Cas9 protein structure. These localized changes enhance the precision of target recognition and reduce off-target binding, thereby maintaining ease of operation while reducing harmful off-target effects through improved specificity at critical functional sites.
3Device complexity
If standard CRISPR/Cas nucleases are used, then simplicity is maintained, but enzymatic activity under optimal conditions is reduced
Solution Approach 1:
The patent applies parameter changes by systematically varying amino acid sequences at specific positions to optimize enzymatic activity parameters such as catalytic efficiency, temperature tolerance, and pH optima. These engineered variants maintain the simple CRISPR/Cas system architecture while enhancing power through improved nuclease activity and stability under diverse physiological conditions.
Data Source
AI summary
The present disclosure provides nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides compositions and system comprising a nuclease comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-1096 and at least one gRNA.