Nucleobase Editors Using Cas9 Nickase for Safe Base Correction
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Solution Overview
Problem
Current CRISPR/Cas9-based genome editing methods for correcting Duchenne muscular dystrophy mutations are prone to safety concerns such as large deletions and chromosomal rearrangements due to double-strand DNA breaks, necessitating precise and safe alternatives for mutation correction.
Innovation Solution
Development of fusion proteins combining Cas9 nickases with nucleotide deaminases, specifically engineered for precise base editing by introducing targeted nucleotide substitutions without DNA cleavage, utilizing recombinant adeno-associated virus (AAV) delivery for in vivo applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9 double-strand DNA break method is used for exon deletion, then dystrophin expression can be restored, but large deletions and chromosomal rearrangements occur causing safety concerns
Solution Approach 1:
The Cas9 nuclease is segmented into two separate nickases, each targeting one strand of the DNA. Instead of creating a double-strand break with one Cas9 protein, two separate Cas9 nickases are used: one nickase targets the forward strand and the other targets the reverse strand. This segmentation prevents the formation of harmful double-strand breaks while still achieving the desired genomic modification through two coordinated single-strand nicks.
Solution Approach 2:
A unique 4-nucleotide overhang intermediate is introduced as a mediator between the two nicking events. The first Cas9 nickase creates a nick with a specific 4-nucleotide overhang, and the second Cas9 nickase is designed to recognize and nick at a position that creates a complementary overhang. This intermediate overhang structure serves as a safe intermediary that guides the repair mechanism to join the exon in-frame without creating large deletions or chromosomal rearrangements.
2Productivity
If CRISPR/Cas9 creates double-strand DNA breaks for mutation correction, then genome editing can be achieved, but unwanted large deletions and chromosomal rearrangement occur
Solution Approach 1:
Instead of using the conventional approach of creating a double-strand break and relying on error-prone non-homologous end joining (NHEJ) repair, the invention inverts the strategy by creating two controlled single-strand nicks that are subsequently repaired by the high-fidelity homology-directed repair (HDR) pathway. This inversion of the breakage-repair paradigm eliminates the harmful effects of DSBs while maintaining editing efficiency.
Solution Approach 2:
The invention converts the potential harm of DNA breaks into a benefit by using controlled single-strand nicks instead of dangerous double-strand breaks. The single-strand nicks are sufficient to trigger the repair machinery but lack the catastrophic effects of DSBs. The 4-nucleotide overhang intermediate further converts what could be a source of error into a precise guide for correct repair, turning a potential harmful byproduct into a beneficial feature that ensures accuracy.
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
Achieves precise correction of disease-associated mutations, restoring dystrophin expression and function in genetic diseases like Duchenne muscular dystrophy, with reduced off-target effects and improved safety profiles.
Implementation Method 1
the Cas9 nickase when in conjunction with a bound guide RNA (gRNA) specifically binds to a target nucleic acid sequence
Implementation Method 2
a nucleotide deaminase, wherein the Cas9 nickase comprises a first amino acid substitution
Data Source
AI summary
The present disclosure relates to nucleobase editors and methods of use thereof.