SCON System for Conditional Gene Knockout

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Solution Overview

Problem

The generation of conditional alleles using CRISPR technology is challenging, particularly due to hypomorphic effects and the complexity of inserting conditional introns in various organisms, which limits their utility in studying essential genes.

Innovation Solution

A genetic element comprising a splice donor site, a recombinase recognition site, a splice branch point, and a splice acceptor site, where the branch point is positioned 10 to 56 nucleotides from the splice acceptor site, enabling a rapid and efficient generation of conditional alleles through CRISPR/Cas9-mediated insertion, known as the Short Conditional intrON (SCON) system.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional conditional knockout approaches using Cre/LoxP system with long intronic cassettes are used, then conditional gene knockout can be achieved, but the long length of the intron causes hypomorphic effects and reduces ease of manufacture

Engineering Contradiction:

Improveconditional knockout functionalityVSAvoidintron length

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention segments the intron into a compact form by removing unnecessary sequences while retaining essential splice sites and branch points. The optimized intron is divided into minimal functional elements: splice donor site, branch point, and splice acceptor site, reducing the overall length from traditional long introns to a compact form that avoids hypomorphic effects.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention changes the length parameter of the intron from traditional long sequences to a optimized compact form (10-56 nucleotides between branch point and acceptor site). This parameter optimization maintains splicing functionality while eliminating hypomorphic effects and improving ease of manufacture.

Inventive Principle:

Principle #35Parameter changes

2Ease of manufacture

If CRISPR/Cas9-mediated insertion of LoxP sites is used to generate cKO alleles, then conditional alleles can be created, but the process remains challenging and complex

Engineering Contradiction:

Improveconditional allele generationVSAvoidCRISPR insertion complexity

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The invention performs preliminary action by pre-designing and pre-optimizing the intron sequence with all necessary splice sites and branch points already in place. This allows the CRISPR/Cas9 system to simply insert the pre-optimized intron into the target gene, significantly simplifying the overall process of conditional allele generation compared to traditional methods.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention optimizes the intron parameters (length, sequence composition, spacing between functional elements) to make the CRISPR insertion process more efficient. The compact intron design with optimized parameters reduces the complexity of the CRISPR-mediated insertion process and improves ease of manufacture.

Inventive Principle:

Principle #35Parameter changes

3Ease of operation

If simple knockout approach is used to study essential genes, then gene function can be investigated, but early developmental lethality occurs

Engineering Contradiction:

Improvegene function studyVSAvoiddevelopmental viability

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The invention introduces dynamics by enabling temporal control of gene knockout through the Cre/LoxP system. The conditional intron allows the gene to remain functional during early development and only be knocked out when Cre recombinase is activated, providing dynamic control over when gene inactivation occurs. This resolves the contradiction by allowing study of essential genes without causing early developmental lethality.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The invention performs preliminary action by inserting the conditional intron into the gene before actual knockout is needed. This allows the gene to function normally during development, and knockout can be triggered later when required, preventing early developmental lethality while enabling gene function studies.

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

SCON allows for conditional knock-out approaches with minimal hypomorphic effects, facilitating easy integration into various organisms, including vertebrates, and enables spatiotemporal control of gene knockout, reducing developmental lethality and improving the study of essential genes.

Implementation Method 1

CRISPR/Cas9-mediated insertion of LoxP sites

Methodology Applied

Scientific EffectCRISPR/Cas9-mediated insertion:

Implementation Method 2

activating recombination at the recombinase recognition sites

Methodology Applied

Scientific EffectRecombinase-mediated recombination:

Implementation Method 3

a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site

Methodology Applied

Scientific EffectSplicing:

Data Source

PatentUS20240229080A1Controlled gene expression methods and means

Publication Date: 2024.07.11 IMBA INSTITUT FUR MOLEKULARE BIOTECH

AI summary

A genetic element including a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site, wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site, and its uses in controlled gene inactivation in a cell is disclosed.