Cas12a Nuclease Engineering for Precise Low Off-Target Editing

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Solution Overview

Problem

Existing CRISPR-Cas editing systems face limitations in efficiency, accuracy, and off-target effects, complicating precise genome editing, especially in commercially relevant applications such as gene replacement.

Innovation Solution

Development of novel CRISPR/Cas12a-based nucleases with altered recognition and a dual gRNA system, which recognize T-rich PAM sequences and create staggered DNA breaks, offering improved editing efficiency and reduced off-target rates.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional CRISPR-Cas editing systems are used, then basic genome editing can be achieved, but editing efficiency is low and off-target effects occur

Engineering Contradiction:

Improveediting efficiencyVSAvoidoff-target effects

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent modifies the Cas12a nuclease by changing specific amino acid parameters at positions 63, 64, and 65 (Q63A, Q64A, Q65A) to alter its recognition specificity and editing parameters, thereby improving editing efficiency while reducing off-target effects

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs a dual gRNA system that divides the guide RNA into two separate components, allowing independent optimization of each gRNA's specificity and binding characteristics to reduce off-target effects while maintaining high editing efficiency

Inventive Principle:

Principle #1Segmentation

2Manufacturing precision

If Cas12a nucleases with altered recognition are developed, then editing precision is improved, but system complexity increases

Engineering Contradiction:

Improvegenome editing precisionVSAvoidsystem complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent achieves improved editing precision by making specific parameter changes to the Cas12a nuclease sequence (amino acid substitutions at positions 63-65) rather than fundamentally redesigning the system, thus maintaining relative simplicity while enhancing precision

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The modified Cas12a nuclease maintains the universal functionality of CRISPR-Cas systems for genome editing while adding enhanced precision through specific amino acid changes, allowing the same basic system to serve multiple editing applications with improved accuracy

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

Enhances genome editing precision and efficiency, reducing off-target effects, and enabling broader applications in various species including humans, by utilizing a CRISPR/Cas12a system with a dual gRNA configuration.

Implementation Method 1

nucleic acid-guided nucleases... recognize T-rich PAM sequences and create staggered DNA breaks

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Data Source

PatentUS20260009009A1Modified nucleases

Publication Date: 2026.01.08 CELYNTRA THERAPEUTICS SA

AI summary

Provided herein are methods and compositions utilizing modified nucleases and/or other components, such as guide nucleic acids and donor templates, for use in a CRISPR system.