CRISPR-Cas Exon Skipping via Splice Site Cleavage

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Solution Overview

Problem

Current genome editing techniques for exon skipping in dystrophin gene editing, such as CRISPR-Cas, face challenges including non-specific cleavage risks, requirement of multiple guide RNAs, and potential side effects due to large deletion sizes and unknown regulatory regions, which hinder efficient and specific induction of exon skipping in Duchenne muscular dystrophy treatment.

Innovation Solution

The method involves using a marker gene with a sequence containing a first and second intron, positioned near the target exon, and designing guide RNA to position CRISPR-Cas cleavage within 80 bases from the splice acceptor or donor site, enhancing exon skipping efficiency and specificity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If two guide RNAs are used to cleave both ends of the target exon, then exon skipping can be induced, but the risk of non-specific cleavage increases and the method complexity increases

Engineering Contradiction:

Improveexon skipping inductionVSAvoidnon-specific cleavage risk

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and targets only the splice acceptor site sequence (2-10 bases immediately upstream of the target exon) rather than requiring cleavage at both ends of the exon. This single-target approach eliminates the need for two guide RNAs and reduces non-specific cleavage risks while maintaining exon skipping induction capability.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of cleaving the exon boundaries directly as in conventional methods, the invention cleaves the splice acceptor site sequence upstream of the exon. This inverted approach disrupts splicing recognition rather than physical exon boundaries, achieving exon skipping with higher specificity.

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If at least several hundred bases are deleted to induce exon skipping, then the exon can be effectively removed, but the risk of unexpected side effects increases due to unknown regulatory regions or miRNA-coding regions

Engineering Contradiction:

Improveexon skipping inductionVSAvoidunexpected side effects

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The invention focuses the cleavage action on a very specific local region (splice acceptor site sequence, 2-10 bases upstream of the exon) rather than deleting large segments. This localized approach ensures exon skipping while preserving distant regulatory elements and miRNA regions that would otherwise be affected by large deletions.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention uses partial action by targeting only the critical splice acceptor site sequence rather than the entire exon or large surrounding regions. This minimal intervention achieves the desired splicing disruption without excessive deletion that could harm other genomic functions.

Inventive Principle:

Principle #16Partial or excessive action

3Productivity

If the splice acceptor sequence is targeted for cleavage, then exon skipping efficiency increases, but the difficulty of designing highly specific guide RNA increases due to similar sequences in many exon sequences

Engineering Contradiction:

Improveexon skipping efficiencyVSAvoidguide RNA design complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The invention performs preliminary analysis to identify unique or highly specific splice acceptor site sequences for each target exon before designing the guide RNA. By pre-selecting optimal target sites within the 2-10 base upstream region that have high specificity, the method achieves both high exon skipping efficiency and easy guide RNA design.

Inventive Principle:

Principle #10Preliminary action

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

This approach increases the efficiency of exon skipping, reduces side effects, and allows for more precise and effective treatment of Duchenne muscular dystrophy by minimizing DNA base deletions required for exon skipping, thereby improving therapeutic outcomes.

Implementation Method 1

CRISPR-Cas genome editing techniques... can bind to a target DNA dependently on a spacer sequence contained in a guide RNA (gRNA or sgRNA), to induce a double-strand DNA break by the action of a Cas nuclease

Methodology Applied

Scientific EffectCRISPR-Cas genome editing:

Implementation Method 2

DNA double-strand break is induced to cause local induction of a DNA repair mechanism through non-homologous recombination (non-homologous end joining, NHEJ)

Methodology Applied

Scientific EffectNon-homologous end joining:

Implementation Method 3

DNA double-strand break is induced to cause local induction of a DNA repair mechanism through non-homologous recombination (non-homologous end joining, NHEJ) or homologous recombination (homology directed repair, HDR)

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentEP3617311B1Method for inducing exon skipping by genome editing

Publication Date: 2024.11.20 KYOTO UNIV

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AI summary

A method of skipping a target exon of a gene of interest in a genome, comprising using CRISPR-Cas and guide RNA, wherein the guide RNA contains a spacer sequence such that the site of cleavage by the CRISPR-Cas is positioned within 80 bases from the splice donor site immediately before the target exon or the splice acceptor site immediately after the target exon.