Cas12a Bridge Helix Variants for DNA Cleavage Selectivity

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Solution Overview

Problem

Current CRISPR-Cas12 proteins face challenges in DNA cleavage specificity, necessitating improved selectivity for precise gene editing applications.

Innovation Solution

Development of variant Cas12a and Cas12b proteins with specific amino acid substitutions in the bridge helix region, such as replacing certain amino acids with proline, to enhance DNA cleavage selectivity, including variants from Francisella tularensis novicida, Lachnospiraceae bacterium, Acidaminococcus sp, and Alicyclobacillus acidoterrestris strains.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type Cas12 proteins are used, then DNA cleavage activity is achieved, but DNA cleavage selectivity is insufficient

Engineering Contradiction:

ImproveDNA cleavage selectivityVSAvoidprotein modification complexity

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent applies local quality by introducing specific amino acid substitutions only in the bridge helix region of the Cas12 protein, rather than modifying the entire protein. This localized modification approach improves DNA cleavage selectivity while minimizing overall protein changes, making the modification process more manageable and less complex.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs parameter changes by systematically varying amino acid residues at specific positions within the bridge helix region. By changing the chemical parameters (amino acid types) at these critical positions, the patent optimizes the protein's DNA binding and cleavage properties, thereby improving selectivity without requiring complete protein redesign.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If amino acid substitutions are introduced in the bridge helix region, then DNA cleavage selectivity is improved, but protein structure stability may be affected

Engineering Contradiction:

ImproveDNA cleavage selectivityVSAvoidprotein structure stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent systematically tests different amino acid substitutions at specific bridge helix positions to identify changes that improve DNA cleavage selectivity while maintaining protein stability. By carefully selecting and testing various amino acid parameters, the patent finds optimal substitutions that achieve the desired selectivity without compromising overall protein structure.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs feedback mechanisms by evaluating the performance of variant Cas12 proteins with different bridge helix substitutions. Through iterative testing and characterization of cleavage activity and selectivity, the patent identifies successful substitutions and uses this information to guide further optimization, ensuring that stability is maintained while improving selectivity.

Inventive Principle:

Principle #23Feedback

Data Source

PatentUS12163166B2Variant CAS12 proteins with improved DNA cleavage selectivity and methods of use

Publication Date: 2024.12.10 THE BOARD OF RGT UNIV OF OKLAHOMA

AI summary

Bridge helix-modified variant Cas12a and Cas12b proteins having improved DNA cleavage selectivity in comparison to wild type versions of the Cas12a and Cas12b proteins, nucleic acids encoding the variant proteins, host cells containing the nucleic acids, and methods of their use.