CRISPR-Cas9 Nickase Complex for Precise Genomic Editing

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Solution Overview

Problem

Current CRISPR-Cas9 systems for gene editing are inefficient in correcting mutations via homology-directed repair (HDR) and often result in non-homologous end-joining (NHEJ), leading to incomplete or incorrect repairs, and have concerns regarding off-target modifications.

Innovation Solution

A complex comprising a guide RNA, a recombinant site-directed nuclease operably linked to a supercharged protein, and a single-stranded donor oligonucleotide (ssODN) is introduced into cells to specifically target and correct mutations by promoting HDR, while minimizing NHEJ and off-target effects.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CRISPR-Cas9 systems are used for gene editing, then genomic sites can be targeted and double-strand breaks can be created, but the efficiency of homology-directed repair (HDR) is low and non-homologous end-joining (NHEJ) predominates

Engineering Contradiction:

ImproveHDR efficiencyVSAvoidrepair accuracy

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent employs a nickase variant of Cas9 (D10A mutation) that creates single-strand breaks instead of double-strand breaks. By using two guide RNAs targeting opposite strands, the system achieves precise control over break formation, promoting HDR over NHEJ. This parameter change in nuclease activity fundamentally shifts the repair pathway preference toward homology-directed repair.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces a donor oligonucleotide as an intermediary template that mediates the repair process. This single-stranded or double-stranded DNA template containing the desired correction sequence facilitates HDR by providing the homology arms needed for strand invasion and repair, thereby increasing both HDR efficiency and repair accuracy.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If CRISPR-Cas9 systems are used for gene editing, then mutations can be targeted, but off-target modifications occur

Engineering Contradiction:

Improvetargeting specificityVSAvoidoff-target effects

Core Design Contradiction:

Manufacturing precisionVSObject-affected harmful factors

Solution Approach 1:

The patent divides the cutting function into two separate guide RNAs targeting opposite strands. Each guide RNA independently binds to its target sequence and directs the nickase to create a single-strand break. This segmentation requires two independent binding events for successful editing, significantly reducing off-target effects while maintaining on-target efficiency.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Instead of using wild-type Cas9 that creates double-strand breaks with one guide RNA, the patent inverts the approach by using nickase Cas9 with two guide RNAs. This reversal of the mechanism transforms the system from high-potency but low-specificity to controlled and highly specific editing.

Inventive Principle:

Principle #13The other way round (Inversion)

3Productivity

If standard CRISPR-Cas9 complexes are used, then genomic editing can be performed, but cell survival rates are reduced

Engineering Contradiction:

Improveediting efficiencyVSAvoidcell survival rate

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent inverts the damage mechanism by using nickases that create single-strand breaks instead of double-strand breaks. Single-strand breaks are much less cytotoxic and allow cells to survive and continue dividing while still enabling efficient HDR when appropriate donor templates are provided, thereby maintaining editing efficiency while dramatically improving cell survival rates.

Inventive Principle:

Principle #13The other way round (Inversion)

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 approach significantly enhances the efficiency of mutation correction, increases the HDR to NHEJ ratio, and ensures high cell survival rates, with specificity demonstrated by whole-genome sequencing showing no off-target modifications in corrected cells.

Implementation Method 1

a guide RNA (gRNA) comprising a first nucleotide sequence that hybridizes to a target DNA in the genome of the cell

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

the site-directed nuclease specifically binds and cleaves the target DNA to create a double stranded break

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Implementation Method 3

a single-stranded donor oligonucleotide (ssODN) that hybridizes to a genomic sequence flanking the double stranded break in the target DNA and integrates into the target DNA to correct a mutation

Methodology Applied

Scientific EffectHomology-directed repair:

Data Source

PatentUS20240117380A1Crispr/cas9 complex for genomic editing

Publication Date: 2024.04.11 THE UAB RESEARCH FOUNDATION INC

AI summary

Provided herein are CRISPR/Cas9 complexes and method of using same.