Mutant Cas9 Proteins Resolving Off-Target Editing Contradictions
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Solution Overview
Problem
Current CRISPR-Cas9 systems suffer from high off-target editing activity, which complicates research and medical applications, and previously developed mutant Cas9 enzymes with reduced off-target activity exhibit reduced on-target activity, limiting their utility, especially when used in ribonucleoprotein (RNP) formats.
Innovation Solution
Development of mutant Cas9 proteins with specific amino acid substitutions, such as at positions N690, F682, L683, K684, and D686, which reduce off-target editing while maintaining high on-target editing activity, even when delivered as RNP complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If mutant Cas9 proteins with reduced off-target activity are developed, then off-target editing activity is reduced, but on-target editing activity is also reduced
Solution Approach 1:
The patent applies parameter changes by systematically varying amino acid substitutions at specific positions (N690, F682, L683, K684, D686) in the Cas9 protein. Each substitution represents a parameter change that selectively reduces off-target binding affinity while preserving on-target cleavage efficiency. The patent identifies optimal substitution patterns that resolve the contradiction between reducing harmful off-target effects and maintaining reliable on-target function.
Solution Approach 2:
The patent applies local quality by introducing specific amino acid substitutions at localized positions within the Cas9 protein structure. Rather than globally modifying the entire protein, the invention targets specific residues (N690, F682, L683, K684, D686) that are critical for distinguishing on-target versus off-target binding. This localized modification approach allows selective reduction of off-target activity while preserving on-target function.
2Productivity
If Cas9 proteins are delivered as RNP complexes, then delivery efficiency is improved, but off-target activity remains high
Solution Approach 1:
The patent combines parameter changes in the Cas9 protein sequence with the RNP delivery format. By incorporating specific amino acid substitutions (N690, F682, L683, K684, D686) into the Cas9 component of the RNP complex, the invention achieves both efficient delivery and reduced off-target activity. The parameter-changed Cas9 protein maintains its ability to form functional RNPs while exhibiting improved specificity.
3Measurement precision
If amino acid substitutions are introduced to reduce off-target activity, then specificity is improved, but enzymatic activity is reduced
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at localized positions within the Cas9 protein structure. Rather than globally modifying the entire protein, the invention targets specific residues (N690, F682, L683, K684, D686) that are critical for distinguishing on-target versus off-target binding. This localized modification approach allows selective reduction of off-target activity while preserving on-target function.
Solution Approach 2:
The patent applies parameter changes by systematically varying amino acid substitutions at specific positions (N690, F682, L683, K684, D686) in the Cas9 protein. Each substitution represents a parameter change that selectively reduces off-target binding affinity while preserving on-target cleavage efficiency. The patent identifies optimal substitution patterns that resolve the contradiction between reducing harmful off-target effects and maintaining reliable on-target function.
Data Source
AI summary
This invention pertains to isolated mutant Cas9 nucleic acids and proteins for use in CRISPR/Cas endonuclease systems and their methods of use. In particular, the invention pertains to an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays a lower ratio of reduced off-target editing activity to on-target editing activity for at least one target site relative to the corresponding ratio of reduced off-site editing activity to on-target editing activity of a wild-type CRISPR/Cas endonuclease system having a WT-Cas9 protein of SEQ ID NO:5.