CRISPR Nickase Gene Knockout for Malignant Transformation Risk
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Solution Overview
Problem
Current methods for targeting the knockout of genes associated with autosomal-dominant disorders like congenital neutropenia are complex, cumbersome, and may introduce novel mutations or malignant transformations, posing safety concerns for clinical applications.
Innovation Solution
A composition comprising two CRISPR endonucleases configured as nickases, along with specific guide RNAs, is used to create single-strand DNA breaks in the regulatory elements of the target gene, ensuring targeted knockout without affecting the coding region, thereby reducing the risk of malignant transformations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for targeted gene knockout are used, then gene knockout can be achieved, but the process becomes complex and may introduce novel mutations or malignant transformations
Solution Approach 1:
The patent divides the gene knockout process into two separate steps: first creating a single-strand break with a nickase, then introducing a repair template. This segmentation avoids the need for double-strand breaks, thereby reducing complexity while maintaining safety by preventing unwanted mutations and malignant transformations that could arise from more aggressive knockout methods
Solution Approach 2:
The patent uses a repair template as an intermediary element that guides the cellular repair mechanism to introduce the desired knockout without relying on error-prone double-strand break repair. This intermediary approach simplifies the process by providing a clear template for the desired outcome while avoiding the harmful effects of direct double-strand cleavage
2Productivity
If double-strand DNA breaks are created to achieve gene knockout, then knockout efficiency is improved, but the risk of malignant transformations and leukemia development increases
Solution Approach 1:
The patent changes the critical parameter of DNA break type from double-strand breaks to single-strand breaks. This parameter change maintains sufficient knockout efficiency by still disrupting the gene's regulatory elements while dramatically reducing the harmful effects associated with double-strand break repair, including malignant transformations and leukemia development
Solution Approach 2:
The patent converts the potentially harmful double-strand break mechanism into a beneficial single-strand break approach. By using nickases that create only single-strand breaks, the method maintains gene disruption capability while eliminating the severe safety risks, effectively turning a harmful mechanism into a safe and effective therapy
3Manufacturing precision
If CRISPR endonucleases are used to create DNA breaks, then targeted gene disruption is achieved, but off-target effects and unintended mutations may occur
Solution Approach 1:
The patent segments the cutting function into two separate nickases, each targeting one strand of the DNA. This segmentation increases precision because both nickases must bind correctly to their respective strands to achieve the desired effect, thereby reducing off-target mutations while maintaining targeted disruption of the gene's regulatory elements
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 allows for a reliable and safe permanent knockout of the target gene, effectively treating autosomal-dominantly inherited disorders like congenital neutropenia without the risks associated with existing methods, such as leukemia development, and maintains the safety profile for clinical use.
Implementation Method 1
A composition comprising two CRISPR endonucleases configured as nickases, along with specific guide RNAs, is used to create single-strand DNA breaks in the regulatory elements of the target gene
Data Source
AI summary
A composition is for the targeted knockout of a gene on double-stranded DNA in a biological cell. A method is for the targeted knockout of a gene on double-stranded DNA in a biological cell. A preparation includes a biological cell prepared in vitro. The biological cell includes a gene on double-stranded DNA, which is knocked-out in a targeted manner. A kit is for the targeted knockout of a gene on double-stranded DNA in a biological cell. Another method is for treating a subject afflicted with a disease associated with a mutated gene. Nucleic acid molecules can be a component of the composition and methods.