Base Editor Exon Skipping Without Double-Strand Breaks
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Solution Overview
Problem
Existing exon skipping methods are either transient or require double-strand breaks in the genome, leading to unpredictable phenotypic outcomes and off-target mutations, while CRISPR-Cas9 gene editing introduces random insertions and deletions.
Innovation Solution
A fusion protein comprising tRNA-specific adenosine deaminases (TadA) domains, a linker, and a RNA-guided DNA endonuclease with nickase activity is used to induce selective exon skipping by contacting DNA target sequences with a single guide RNA (sgRNA) and the fusion protein, without causing double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 introduces double-strand breaks to achieve exon skipping, then exon skipping is achieved, but off-target mutations and unpredictable phenotypic outcomes occur
Solution Approach 1:
The Cas9 endonuclease is divided into two separate nickase components, each capable of cutting only one DNA strand. These two nickases work together to produce the desired exon skipping effect while avoiding the harmful double-strand breaks that cause off-target mutations
Solution Approach 2:
A fusion protein is introduced as an intermediary that combines the two nickase components with a linker sequence. This fusion protein mediates the coordinated action of the nickases to achieve precise exon skipping while minimizing off-target effects through controlled single-strand cutting
2Manufacturing precision
If antisense oligonucleotides are used for exon skipping, then exon skipping is achieved, but the effect is transient requiring repeated injections
Solution Approach 1:
The base editor system performs preliminary permanent modification of the DNA sequence at the target exon region before the therapeutic effect is needed. This preliminary genetic modification ensures long-lasting exon skipping without requiring repeated administrations
Solution Approach 2:
The transient mechanical action of antisense oligonucleotides is replaced with a permanent biochemical modification system using base editors. This substitution transforms the temporary physical blocking mechanism into a lasting genetic modification that persists without repeated doses
3Duration of action of stationary object
If base editors are used to achieve permanent exon skipping, then lasting modifications are achieved, but the system complexity increases
Solution Approach 1:
The base editor system is designed as a universal platform that can target multiple different exons and genes using the same core components. The sgRNA provides flexibility to redirect the system to different targets, reducing overall system complexity through multi-functionality
Solution Approach 2:
Rather than attempting to design completely separate systems for each therapeutic application, the invention uses a partial system approach where the core base editor machinery remains constant and only the sgRNA needs to be modified for different targets, simplifying the overall system while achieving permanent modifications
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 permanent and precise exon skipping with reduced off-target effects, providing a therapeutic tool for diseases like Huntington's and Duchenne Muscular Dystrophy.
Implementation Method 1
tRNA-specific adenosine deaminases (TadA) domains
Implementation Method 2
RNA-guided DNA endonuclease having nickase activity protein
Data Source
AI summary
The disclosure provides a versatile method termed CRISPR-SKIP that utilizes cytidine and/or adenine deaminase base editors to program exon skipping by mutating target DNA bases within splice acceptor sites and/or splice enhancer sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.