Chimeric Nuclease Gene Targeting Efficiency

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current gene targeting techniques in mammalian cells have low efficiency, with most introduced genetic changes occurring at non-homologous sites rather than replacing mutated genes, limiting their applicability for therapeutic and experimental purposes, including gene therapy.

Innovation Solution

The use of chimeric nucleases, comprising a DNA binding domain and a cleavage domain, is introduced into cells along with a repair substrate to specifically alter genomic sequences, enhancing the frequency of targeted gene modifications by creating double-stranded breaks and facilitating homologous recombination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional gene targeting methods are used in mammalian cells, then genetic changes can be introduced, but the efficiency is extremely low with most changes occurring at non-homologous sites

Engineering Contradiction:

Improvegene targeting efficiencyVSAvoidtargeting accuracy

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The chimeric nuclease is divided into two functional domains: a DNA binding domain (zinc fingers) that recognizes and binds to the target sequence, and a cleavage domain (FokI restriction enzyme) that creates the double-stranded break. This segmentation allows the binding and cleavage functions to be optimized independently, enabling precise targeting while maintaining high efficiency

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The zinc finger DNA binding domain acts as an intermediary that mediates the interaction between the nuclease and the specific target sequence. By designing zinc fingers that recognize specific DNA sequences, the system directs the cleavage activity precisely to the intended genomic location, thereby improving targeting accuracy without sacrificing productivity

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If genes are introduced into mammalian cells using conventional methods, then genetic material can be delivered, but integration occurs primarily at non-homologous sites rather than replacing mutated genes

Engineering Contradiction:

Improvegene integration capabilityVSAvoidintegration site specificity

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The invention changes the key parameter of DNA integration by creating controlled double-stranded breaks at specific locations. This forces the cell's repair mechanisms to act at defined sites rather than allowing random integration, thereby achieving both versatility in gene delivery and precision in integration site selection through homologous recombination

Inventive Principle:

Principle #35Parameter changes

3Productivity

If the rate of gene targeting is extremely low in current cell types, then conventional gene therapy approaches are limited, but developing new techniques is needed to increase efficiency

Engineering Contradiction:

Improvegene targeting rateVSAvoidmethod complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The chimeric nuclease combines two previously separate biological components (zinc finger DNA-binding proteins and FokI restriction enzyme) into a single fusion protein. This composite structure integrates DNA recognition and cleavage functions, dramatically increasing gene targeting rate while the modular design keeps the overall method complexity manageable

Inventive Principle:

Principle #40Composite materials

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 significantly increases the efficiency of gene targeting, achieving rates of up to 3-5% in mammalian cells, making it potentially therapeutic for diseases like severe combined immunodeficiency, sickle cell disease, and hemophilia, and providing a tool for experimental manipulation of the genome.

Implementation Method 1

introducing a repair substrate into the cell, wherein said repair substrate comprises: (i) a nucleic acid sequence that is substantially identical to regions on one or both sides of the target sequence; and (ii) a nucleic acid sequence which changes the target sequence upon recombination between the repair substrate and the target sequence

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS9447434B2Use of chimeric nucleases to stimulate gene targeting

Publication Date: 2016.09.20 CALIFORNIA INST OF TECH

AI summary

Gene targeting is a technique to introduce genetic change into one or more specific locations in the genome of a cell. For example, gene targeting can introduce genetic change by modifying, repairing, attenuating or inactivating a target gene or other chromosomal DNA. In one aspect, this disclosure relates to methods and compositions for gene targeting with high efficiency in a cell. This disclosure also relates to methods of treating or preventing a genetic disease in an individual in need thereof. Further disclosed are chimeric nucleases and vectors encoding chimeric nucleases.