CRISPR Nuclease Variant Compositions for Stable RNA-Guide Binding
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Solution Overview
Problem
Existing CRISPR-Cas systems face challenges in achieving enhanced enzymatic activity, binding activity, binding specificity, and stability, which are crucial for efficient nucleic acid targeting and editing.
Innovation Solution
The development of a variant polypeptide that comprises alterations relative to a parent polypeptide, such as SEQ ID NO: 3, resulting in enhanced enzymatic activity, binding activity, binding specificity, and stability, when complexed with an RNA guide and a target nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a CRISPR nuclease system is used for nucleic acid targeting, then binding activity is achieved, but enzymatic activity and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (such as positions 509, 511, 521, 535, 198, 527, 580, 359, 381, 354, 380, 383, 367, 507, 595, 531, 129, 615, 618, 578, 158, 476, 371, 136, 220, 78, 141, 240, 277, 165, and 374) to optimize the balance between stability and enzymatic activity. The variant nucleases exhibit enhanced stability while maintaining or improving enzymatic function compared to parent nucleases.
Solution Approach 2:
The patent applies local quality by making specific localized amino acid substitutions at particular positions within the nuclease structure rather than uniform modifications. Each position has been identified to contribute specifically to either stability or enzymatic activity, allowing localized optimization of protein properties while preserving overall structure and function.
2Measurement precision
If binding specificity is enhanced through variant modifications, then off-target effects are reduced, but binding activity may be compromised
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues that influence both binding specificity and binding activity. The variants demonstrate improved binding specificity with reduced off-target effects while maintaining robust binding activity, achieving a favorable balance between these two critical parameters through systematic amino acid substitution.
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
The variant polypeptide exhibits improved performance in binding to RNA guides and target nucleic acids, leading to enhanced enzymatic activity and stability, thereby improving the efficiency and specificity of nucleic acid targeting and editing processes.
Implementation Method 1
the variant polypeptide is capable of binding to an RNA guide and a target nucleic acid
Implementation Method 2
the variant polypeptide or a complex comprising the variant polypeptide exhibits enhanced enzymatic activity
Data Source
AI summary
The present disclosure relates to variant polypeptides, methods of preparing the variant polypeptides, processes for characterizing the variant polypeptides, compositions and cells comprising the variant polypeptides, and methods of using the variant polypeptides. The disclosure further relates to complexes comprising the variant polypeptides, methods of producing the complexes, processes for characterizing the complexes, cells comprising the complexes, and methods of using the complexes.