Engineered CRISPR-Cas9 Nucleases Specificity

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

CRISPR-Cas9 nucleases face challenges in specificity, leading to undesired off-target mutations due to potential excessive energy for recognizing target DNA sites, resulting in cleavage of mismatched sites.

Innovation Solution

Engineering Cas9 variants by introducing alanine substitutions in residues that interact with the DNA backbone, such as N497, R661, and Q695, to reduce non-specific binding affinity, thereby enhancing target specificity while maintaining on-target activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Cas9 binding affinity for DNA is increased to improve target recognition, then on-target activity is enhanced, but off-target effects increase due to excessive binding strength

Engineering Contradiction:

Improveon-target activityVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies parameter changes by systematically mutating specific amino acid residues (N497, R661, Q695, Q926, D1135) that contact the DNA backbone, altering the binding affinity parameters of Cas9 to achieve optimal specificity while maintaining on-target activity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies local quality by making targeted mutations at specific positions within the Cas9 protein structure that interact with DNA, rather than uniformly modifying the entire protein. This localized modification approach allows precise control over binding characteristics at the DNA interaction interface

Inventive Principle:

Principle #3Local quality

2Object-affected harmful factors

If Cas9 binding affinity for DNA is reduced to decrease off-target effects, then target specificity improves, but on-target activity decreases

Engineering Contradiction:

Improveoff-target effectsVSAvoidon-target activity

Core Design Contradiction:

Object-affected harmful factorsVSReliability

Solution Approach 1:

The patent optimizes binding parameters by testing multiple mutation combinations (single, double, triple, quadruple mutants) to find the precise parameter set that reduces off-target effects while preserving sufficient on-target activity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies partial action by introducing a limited number of specific mutations (rather than comprehensive modifications) to achieve the desired balance between specificity and activity, using the minimal necessary changes to resolve the contradiction

Inventive Principle:

Principle #16Partial or excessive action

Data Source

PatentUS10633642B2Engineered CRISPR-Cas9 nucleases

Publication Date: 2020.04.28 THE GENERAL HOSPITAL CORP

AI summary

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.