Sequential Genome Editing Without DNA Templates
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Solution Overview
Problem
Current gene editing technologies, such as CRISPR/Cas, face challenges in achieving efficient site-specific base substitution without introducing foreign DNA fragments, particularly in cell therapy and long-term plant breeding, due to low editing efficiency and safety concerns related to off-target effects.
Innovation Solution
A method involving sequential DNA breaks in an organism's genome, where subsequent breaks are generated based on new sequences formed from previous repair events, allowing for programmed sequential cutting and editing without an artificial DNA template, using CRISPR/Cas systems and targeted nucleases to create site-specific mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If sequence-specific nucleases are used to generate DNA double-strand breaks for site-specific editing, then editing precision is improved, but base substitution efficiency deteriorates
Solution Approach 1:
The invention divides the base substitution process into multiple sequential DNA break events rather than attempting single-step substitution. Each break event is targeted at a specific position, and the cumulative effect of multiple breaks achieves the desired base substitution while maintaining high precision at each step.
Solution Approach 2:
The method performs preliminary DNA breaks at positions adjacent to the target base before the actual substitution occurs. These preliminary breaks create intermediate sequences that facilitate the final base substitution, effectively preparing the genomic site for the desired modification.
2Adaptability or versatility
If exogenous DNA fragments are introduced as repair templates to achieve base substitution, then editing capability is improved, but biological safety deteriorates
Solution Approach 1:
The invention extracts and removes the exogenous DNA template component from the base substitution process. By relying solely on the organism's endogenous repair mechanisms without introducing foreign DNA templates, the method maintains editing capability while eliminating the biological safety concerns associated with transgenic elements.
Solution Approach 2:
The method enables the organism to perform base substitution using its own endogenous repair pathways without requiring external DNA templates. The cell's natural repair machinery is harnessed to achieve the desired genetic modification, making the system self-sufficient and avoiding safety issues related to exogenous DNA introduction.
3Productivity
If multiple DNA breaks are sequentially generated to achieve base substitution, then base substitution efficiency is improved, but device complexity deteriorates
Solution Approach 1:
The invention uses a universal nuclease system (such as CRISPR-Cas9) that can be programmed to create multiple DNA breaks at different positions through simple guide RNA design. This multi-functional approach allows the same core machinery to perform multiple break events, achieving complex base substitution through a unified platform rather than requiring different tools for each step.
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 enables efficient site-specific base substitution, deletion, and insertion mutations, enhancing the editing capabilities of CRISPR/Cas systems and reducing biological safety concerns, thereby facilitating precise genetic modifications in organisms.
Implementation Method 1
Sequence-specific nucleases are programmable nucleases that can generate DNA double-strand breaks (DSBs) at specific sites in genome
Implementation Method 2
adding an exogenous DNA fragment as a repair template to initiate a homologous recombination repair pathway
Implementation Method 3
by introducing a targeted fragmentation into the genome of recipient organism and causing spontaneous repair
Data Source
AI summary
The present invention pertains to the technical field of genetic engineering, and specifically relates to a method for generating a site-specific mutation in an organism in the absence of an artificial DNA template and a use thereof. The method comprises the following steps: sequentially generating two or more DNA breaks at a specific site in a genome of an organism and spontaneously repairing them respectively, wherein a later DNA break is generated based on a new sequence generated from a previous DNA break repair. In the present invention, a new target is designed based on a sequence formed by a new repair event generated by sequential editing, and thus, mutations may be sequentially formed for multiple times at a specific site in the genome, which greatly enrich the types of repair events after DNA breaks, and realize new base substitution, deletion and insertion mutations that cannot be obtained in a single gene editing.