Engineered SpCas9 Variants for Higher On-Target Genome Editing

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Solution Overview

Problem

Existing CRISPR technologies face challenges in achieving moderate-to-high levels of on-target editing, limiting the effectiveness and applicability of genome editing applications.

Innovation Solution

Development of engineered SpCas9 variants with specific mutations at key positions to enhance on-target activity, including combinations of mutations that improve editing efficiency and specificity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type SpCas9 is used for genome editing, then the system is simple and easy to implement, but the on-target editing activity is insufficient

Engineering Contradiction:

Improveon-target editing activityVSAvoidprotein structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies parameter changes by systematically mutating specific amino acid residues in the SpCas9 protein structure. Multiple point mutations at defined positions (such as R221K, N394K, L1245V, E1243K, E1253K) are introduced to enhance on-target editing activity while maintaining the overall protein architecture and function.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates composite protein structures by combining multiple beneficial mutations within the SpCas9 framework. The engineered variants integrate several amino acid substitutions that work synergistically to improve editing efficiency, resulting in a composite enzyme with enhanced properties compared to wild-type SpCas9.

Inventive Principle:

Principle #40Composite materials

2Productivity

If multiple mutations are introduced to enhance editing efficiency, then on-target activity improves, but the risk of off-target effects may increase

Engineering Contradiction:

Improveediting efficiencyVSAvoidoff-target effects

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by making targeted, position-specific mutations at precisely defined locations within the SpCas9 protein. Each mutation is placed at a specific residue position (e.g., R221K, N394K) where it can locally enhance binding affinity or catalytic activity without disrupting the overall specificity mechanisms of the enzyme.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs feedback through systematic evaluation and optimization of mutation combinations. Multiple variant configurations are tested and compared to identify the optimal set of mutations that maximizes on-target activity while maintaining or improving specificity, allowing iterative refinement of the enzyme properties.

Inventive Principle:

Principle #23Feedback

Data Source

PatentUS20250382595A1CRISPR-Cas Enzymes with Enhanced On-Target Activity

Publication Date: 2025.12.18 THE GENERAL HOSPITAL CORP

AI summary

Engineered versions of Streptococcus pyogenes Cas9 (SpCas9) and SpCas9 variants that have improved on-target editing capabilities, and methods of use thereof.