Engineered Cas12i Nuclease Mutations for Gene Editing Efficiency
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Solution Overview
Problem
Current CRISPR-Cas systems for genome editing have limited gene editing efficiency, necessitating improved methods for efficient genome editing across multiple loci.
Innovation Solution
Engineered Cas12i nuclease with specific mutations, including replacing amino acids interacting with PAM, opening double-stranded DNA, RuvC domain, and DNA-RNA double helix with positively charged or aromatic amino acids, and flexible region modifications to enhance catalytic activity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current CRISPR-Cas systems are used for genome editing, then genome editing can be performed, but gene editing efficiency is limited
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (E176R, K238R, T447R, E563R) in the Cas12i nuclease to alter its biochemical properties. These mutations change the charge distribution and interaction interfaces of the protein, thereby improving its catalytic activity and gene editing efficiency while maintaining target specificity
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (176, 238, 447, 563) rather than global modifications. Each mutation is strategically placed to optimize local interactions with DNA substrates and PAM sequences, enhancing overall editing efficiency without compromising other functional regions of the enzyme
2Productivity
If amino acid replacements are made to improve catalytic activity, then gene editing efficiency increases, but protein structure stability may be affected
Solution Approach 1:
The patent carefully selects amino acid substitutions that change local chemical properties (e.g., charge, hydrophobicity) to enhance catalytic activity. The mutations E176R, K238R, T447R, and E563R were chosen to optimize electrostatic interactions with DNA while preserving the overall protein fold and stability through rational design
3Adaptability or versatility
If CRISPR-Cas systems are used for multi-locus editing, then broad genome editing capability is achieved, but editing efficiency at multiple loci remains limited
Solution Approach 1:
The engineered Cas12i nuclease with multiple amino acid substitutions exhibits enhanced catalytic efficiency and processivity, enabling effective simultaneous editing at multiple genomic loci. The improved enzyme maintains high activity across diverse target sequences and PAM variants, making multi-locus editing practical and efficient
Data Source
AI summary
Provided is an engineered Cas12i nuclease, which comprises one or more of the following mutations based on a reference Cas12i nuclease: (1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positively charged amino acids; and/or (2) replacing one or more amino acids involved in opening the double strands of DNA, in the reference Cas12i nuclease with amino acids with aromatic rings; and/or (3) replacing one or more amino acids, which interact with a single-stranded DNA substrate and are located in an RuvC domain in the reference Cas12i nuclease, with positively charged amino acids; and/or (4) replacing one or more amino acids interacting with a DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids; and/or (5) replacing one or more polar or positively charged amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with hydrophobic amino acids. The engineered Cas12i nuclease has significantly improved gene editing efficiency and/or significantly reduced off-target phenomenon compared with the reference Cas12i nuclease.