Engineered Cas9 Variants for AAV Delivery and High-Fidelity Editing
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Solution Overview
Problem
Existing Cas9 proteins are too large for effective delivery via methods like AAV and have low activity, limiting their suitability for therapeutic genome editing applications.
Innovation Solution
Engineering Cas9 proteins with specific amino acid modifications, such as substitutions at positions 37, 39, 46, 47, 54, 57, 59, 61, 94, 315, 501, 508, 556, 566, 911, 912, 978, 980, 1073, 1091, 1122, 1160, 1161, 1164, 1206, 1226, and 1229, to enhance endonuclease activity and fidelity, and incorporating nuclear localization signals (NLS) for improved delivery and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type Cas9 proteins are used, then they can perform genome editing functions, but their large size prevents effective delivery via AAV and their activity is insufficient for therapeutic applications
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (37, 39, 46, 47, 54, 57, 59, 61, 94, 315, 501, 508, 556, 566, 911, 912, 978, 980, 1073, 1091, 1122, 1160, 1161, 1164, 1206, 1226, and 1229) to optimize the Cas9 protein's endonuclease activity while maintaining a compact size suitable for AAV delivery
Solution Approach 2:
The patent creates a composite structure by combining multiple amino acid modifications with nuclear localization signals (NLS) to generate a hybrid Cas9 variant that integrates enhanced enzymatic activity, proper cellular localization, and reduced size into a single functional protein
2Reliability
If Cas9 proteins are engineered with multiple amino acid modifications, then endonuclease activity and fidelity are enhanced, but the complexity of protein engineering increases
Solution Approach 1:
The patent systematically changes specific amino acid parameters at predetermined positions to enhance fidelity while maintaining manageable engineering complexity through focused mutagenesis rather than random screening
3Weight of moving object
If Cas9 proteins are modified to reduce size for AAV delivery, then delivery efficiency improves, but endonuclease activity may be compromised
Solution Approach 1:
The patent optimizes the balance between size and activity by selecting specific amino acid positions for modification that reduce protein mass while preserving or enhancing catalytic function through strategic residue changes
Data Source
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AI summary
The disclosure relates to a Cas9 protein comprising a polypeptide sequence having at least 95% identity, such as 97%, 98% or 99%, to SEQ ID NO: 1, wherein said polypeptide sequence comprises an amino acid modification at one or more positions relative to SEQ ID NO: 1: 37, 39, 46, 47, 54, 57, 59, 61, 94, 315, 501, 508, 556, 566, 911, 912, 978, 980, 1073, 1091, 1122, 1160, 1161, 1164, 1206, 1226 and/or 1229, CRISPR-Cas systems and methods for providing site-specific modification of a target sequence in a eukaryotic cell.