Blunt-Ended CRISPR Editing for Splice Site Correction

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Solution Overview

Problem

Current gene editing technologies, such as HDR, are inefficient in post-mitotic tissues like skeletal muscles for correcting splice site mutations, and existing strategies for neuromuscular disorders like MDC1A are limited due to the large size of the LAMA2 gene and lack of redundant regions, hindering therapeutic effectiveness.

Innovation Solution

A genome editing system that generates blunt-ended DNA breaks in a sequence-specific manner to excise and join DNA ends in introns with splice donor site mutations, restoring functional splice sites through a homology-directed repair-independent mechanism, specifically using Cas9 nucleases and guide RNAs to target and correct mutations in the LAMA2 gene.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If homology-directed repair (HDR) is used to correct splice site mutations, then precise genomic modification can be achieved, but editing efficiency is extremely low in post-mitotic tissues such as skeletal muscles

Engineering Contradiction:

Improvegenomic modification precisionVSAvoidediting efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The invention changes the DNA break end configuration from sticky ends (standard CRISPR/Cas9) to blunt ends. This parameter change enables the use of non-homologous end joining (NHEJ) pathway instead of HDR, achieving high editing efficiency in post-mitotic tissues while maintaining precision through careful design of the blunt-ended breaks to exclude only the mutant nucleotide

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention substitutes the HDR repair mechanism with NHEJ repair mechanism. By inducing blunt-ended DNA breaks rather than sticky-ended breaks, the cellular repair machinery naturally uses NHEJ instead of HDR, achieving high efficiency in non-dividing cells where HDR is ineffective

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Reliability

If standard CRISPR/Cas9 with sticky-ended breaks is used, then homology-directed repair can be promoted, but the requirement for homology templates and cell division reduces therapeutic utility in post-mitotic tissues

Engineering Contradiction:

ImproveHDR pathway reliabilityVSAvoidtherapeutic applicability

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The invention changes the break end configuration parameter from sticky ends to blunt ends. This eliminates the requirement for homology templates and cell division, enabling direct application in post-mitotic tissues like skeletal muscles and neurons without needing external DNA templates or relying on cell proliferation

Inventive Principle:

Principle #35Parameter changes

3Reliability

If the LAMA2 gene is reintroduced via standard gene therapy, then LAMA2 expression can be restored, but the large size of the gene and lack of redundant regions prevents miniaturization strategies

Engineering Contradiction:

ImproveLAMA2 expression restorationVSAvoidgene therapy complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

Instead of reintroducing the entire large LAMA2 gene or using miniaturization strategies that require redundant regions, the invention extracts and corrects only the specific mutant nucleotide causing the splice site defect. This targeted approach restores LAMA2 expression without requiring gene replacement or complex miniaturization

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention segments the problem by focusing only on the specific mutant nucleotide rather than the entire gene. By using blunt-ended CRISPR to target and excise only the defective nucleotide, the approach simplifies the therapeutic intervention from whole-gene replacement to precise nucleotide correction

Inventive Principle:

Principle #1Segmentation

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 effectively restores splice site recognition and LAMA2 expression in skeletal muscles, improving muscle pathology and function in MDC1A models, demonstrating potential for treating neuromuscular disorders by correcting disease-causing mutations in post-mitotic tissues.

Implementation Method 1

at least one nuclease that generates blunt-ended DNA breaks in a sequence-specific manner, wherein the genome editing system is configured to form a first and a second blunt-ended double strand break in an intron

Methodology Applied

Scientific EffectDNA double strand break:

Implementation Method 2

joining DNA ends flanking the excised segment of the intron to constitute a functional donor splice site through a homology-directed repair-independent mechanism

Methodology Applied

Scientific EffectNon-homologous end joining:

Data Source

PatentEP3652310B1Gene editing system for correcting splicing defects

Publication Date: 2023.05.03 HOSPITAL FOR SICK CHILDREN

Figure 1a~1c
Figure 1d~1f
Figure 1g~1h

AI summary

The present disclosure provides genome editing systems, compositions and methods. The genome editing system comprises at least one nuclease that generates blunt-ended DNA breaks in a sequence-specific manner, wherein the genome editing system is configured to form a first and a second blunt-ended double strand break in an intron of a gene, wherein the intron comprises a splice donor site mutation that alters splice site recognition, thereby excising a segment of the intron and simultaneously joining DNA ends flanking the excised segment of the intron to constitute a functional donor splice site.