Cas12i2 Variant Polypeptide Stability and Specificity
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Solution Overview
Problem
Current CRISPR-Cas systems face limitations in terms of specificity and stability of complex formation with RNA guides, leading to off-target binding and reduced enzymatic activity across varying temperatures and pH conditions.
Innovation Solution
A variant Cas12i2 polypeptide with specific sequence modifications, such as those listed in SEQ ID NOs: 3 to 146 and 495 to 512, forms a more stable and specific complex with RNA guides, enhancing binding affinity and stability across a range of temperatures and pH conditions, while maintaining or increasing enzymatic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type Cas12i2 polypeptide is used, then the system is simple and easy to obtain, but the complex formation with RNA guide is unstable and specificity is reduced
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues at defined positions (e.g., positions 581, 624, 626, 835, 836, 868, 879, 911, 1926, 1020, 1030, 1035, and 1046) in the Cas12i2 polypeptide sequence. These targeted modifications alter the structural and functional parameters of the polypeptide to enhance RNA guide binding stability and specificity while maintaining manageable sequence complexity through precise, localized changes rather than comprehensive redesign.
2Measurement precision
If wild-type Cas12i2 polypeptide is used, then the sequence is simple, but off-target binding occurs and on-target specificity is reduced
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at particular positions within the polypeptide sequence that are critical for RNA guide interaction. For example, modifications at positions 581, 624, 626, 835, 836, 868, 879, 911, 1926, 1020, 1030, 1035, and 1046 are strategically placed in regions responsible for RNA binding and guide recognition. These localized changes enhance on-target specificity and reduce off-target binding without requiring complex modifications throughout the entire sequence.
3Reliability
If wild-type Cas12i2 polypeptide is used, then the structure is simple, but enzymatic activity is reduced across varying temperatures and pH conditions
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues that influence the polypeptide's structural stability and catalytic function under varying environmental conditions. The substitutions at positions 581, 624, 626, 835, 836, 868, 879, 911, 1926, 1020, 1030, 1035, and 1046 alter local structural parameters and intermolecular interactions, enabling the Cas12i2 polypeptide to maintain consistent enzymatic activity across a broader range of temperatures and pH levels while introducing only targeted, manageable sequence complexity.
Data Source
AI summary
The present invention relates to variant Cas12i2 polypeptides, methods of producing the variant Cas12i2 polypeptides, processes for characterizing the variant Cas12i2 polypeptides, cells comprising the variant Cas12i2 polypeptides, and methods of using the variant Cas12i2 polypeptides. The invention further relates to complexes comprising the variant Cas12i2 polypeptides, methods of producing the complexes, processes for characterizing the complexes, cells comprising the complexes, and methods of using the complexes.