Cas9 Bridge Helix Variants for DNA Cleavage Selectivity

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Solution Overview

Problem

Cas9's primary drawback is off-target DNA cleavage due to its tolerance of mismatches between the sgRNA-guide and the target DNA, leading to reduced DNA cleavage selectivity and increased off-target effects.

Innovation Solution

Variant Cas9 proteins with amino acid substitutions in the bridge helix (BH) region, such as proline substitutions at positions L64 and K65, enhance DNA cleavage selectivity by improving the protein's ability to discriminate against mismatched DNA substrates.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Cas9 protein is used for DNA targeting, then DNA cleavage activity is achieved, but off-target DNA cleavage occurs due to tolerance of mismatches

Engineering Contradiction:

ImproveDNA cleavage selectivityVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies parameter changes by substituting amino acids at specific positions (K848, N864, and/or K873) in the HNH domain of Cas9 protein. These parameter changes in the protein structure modify the DNA binding interface to increase stringency for mismatch discrimination, thereby improving DNA cleavage selectivity and reducing off-target effects while maintaining on-target activity.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If Cas9 tolerates mismatches between sgRNA-guide and target DNA, then broader target compatibility is achieved, but DNA cleavage precision is reduced

Engineering Contradiction:

Improvetarget compatibilityVSAvoidDNA cleavage precision

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent modifies the HNH domain parameters by substituting amino acids at positions K848, N864, and/or K873. These changes adjust the DNA binding interface properties to enhance mismatch discrimination capability, thereby improving DNA cleavage precision while maintaining appropriate target compatibility through controlled stringency.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS11124783B2Variant CAS9 proteins with improved DNA cleavage selectivity

Publication Date: 2021.09.21 THE BOARD OF RGT UNIV OF OKLAHOMA

AI summary

Bridge helix-modified variant Cas9 proteins having improved DNA cleavage selectivity in comparison to wild type versions of the Cas9 proteins, nucleic acids encoding the variant proteins, host cells containing the nucleic acids, and methods of their use.