Cas9 Nickase Nucleobase Editors for DMD Mutation Correction
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Solution Overview
Problem
Current CRISPR/Cas9-based genome editing methods for correcting mutations in Duchenne muscular dystrophy (DMD) pose safety concerns due to the risk of double strand DNA breaks leading to unwanted large deletions and chromosomal rearrangements.
Innovation Solution
Development of fusion proteins comprising a Cas9 nickase with nucleotide deaminases, specifically engineered for precise base editing by introducing targeted nucleotide substitutions, such as adenine deaminases, to correct disease-associated mutations without inducing DNA breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9-based genome editing is used to correct mutations, then mutation correction capability is improved, but safety deteriorates due to double strand DNA breaks causing large deletions and chromosomal rearrangements
Solution Approach 1:
The patent extracts and removes the harmful double strand break function from the Cas9 enzyme by using a Cas9 nickase variant that only cuts one DNA strand. This eliminates the harmful effects of DSBs while preserving the beneficial target-specific DNA cutting capability needed for mutation correction.
Solution Approach 2:
The patent changes the cutting parameter of Cas9 from double strand break to single strand nick by using a nickase variant. This parameter change maintains the ability to target and cut specific DNA sequences while eliminating the harmful effects associated with double strand breaks.
2Manufacturing precision
If Cas9 nickase with nucleotide deaminases is used for base editing, then precision is improved, but device complexity increases due to fusion protein construction
Solution Approach 1:
The patent merges the Cas9 nickase domain with the nucleotide deaminase domain into a single fusion protein. This combination allows the editor to simultaneously perform DNA nicking and base conversion functions, improving precision while the modular design keeps the complexity manageable.
Solution Approach 2:
The fusion protein combines multiple functions (DNA binding, nicking, and base editing) into a single molecular entity, allowing one protein to perform what would otherwise require multiple separate components, thus improving precision without proportionally increasing complexity.
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
The engineered Cas9 nickase with nucleotide deaminases effectively and precisely corrects genetic mutations, restoring dystrophin expression and function in DMD models, improving muscle function and reducing fibrosis, while minimizing off-target effects.
Implementation Method 1
the nucleotide deaminase is a cytidine deaminase or an adenine deaminase
Data Source
AI summary
The present disclosure relates to nucleobase editors and methods of use thereof. Disclosed herein are fusion proteins, systems, and compositions for editing disease-associated mutations and methods of use thereof. In some aspects, disclosed herein is a fusion protein comprising a Cas9 nickase and a nucleotide deaminase, wherein the Cas9 nickase comprises a first amino acid substitution at a position selected from the group consisting of 262, 324, 409, 480, 543, 694, and 1219 when compared to SEQ ID NO: 11, and wherein the Cas9 nickase comprises a second amino acid substitution at a position selected from the group consisting of 1111, 1135, 1218, 1219, 1322, 1335, and 1337 when compared to SEQ ID NO: 11.