In Vivo Gene Creation via Multi-Site Genome Recombination
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Solution Overview
Problem
Current gene editing tools, such as CRISPR/Cas9, primarily focus on editing internal elements of a single gene, leading to frameshift mutations and function loss, making it difficult to generate new genes or up-regulate gene expression without foreign DNA templates, which are subject to strict regulatory procedures.
Innovation Solution
A method involving simultaneous generation of DNA double-strand breaks at specific sites in an organism's genome, followed by non-homologous end joining or homologous repair to create new combinations of genetic elements or protein domains, allowing for the in vivo creation of new genes without an artificial DNA template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If gene editing tools like CRISPR/Cas9 are used to edit internal elements of a single gene, then site-specific mutations can be generated, but new genes cannot be created and gene expression cannot be up-regulated
Solution Approach 1:
The method divides the genome into multiple targetable segments by designing multiple guide RNAs that bind to different locations. By inducing double-strand breaks at multiple specific sites simultaneously, the method enables recombination of different gene elements (promoters, coding regions, terminators) to create new gene combinations without requiring foreign DNA templates.
Solution Approach 2:
Instead of using foreign DNA templates to introduce new genes (conventional transgenic approach), this method inverts the approach by using the organism's own genome elements. Multiple guide RNAs target endogenous gene sequences, and the cell's repair mechanisms naturally recombine these elements to generate new genes, thereby avoiding regulatory restrictions on transgenic crops.
2Adaptability or versatility
If foreign DNA templates are used to assemble new genes in vitro, then new genes can be introduced, but strict regulatory procedures apply similar to transgenic crops
Solution Approach 1:
The method employs the cell's own DNA repair mechanisms (non-homologous end joining or homologous recombination) to assemble new gene combinations. By delivering multiple guide RNAs and nucleases, the system allows the cell to perform the assembly operation itself using endogenous gene elements, eliminating the need for in vitro gene construction and foreign DNA transformation, thus avoiding transgenic regulatory procedures.
3Productivity
If CRISPR/Cas9 is used for gene knockout, then existing genes can be mutated, but new genes with new functions cannot be generated
Solution Approach 1:
The method combines multiple guide RNA-mediated cleavage events with the cell's DNA repair machinery to achieve gene fusion or promoter swapping. By simultaneously targeting multiple sites within or between genes, the method merges different gene elements (e.g., promoter of gene A with coding region of gene B) to create chimeric genes with novel functions, thereby extending gene editing from simple knockout to de novo gene creation.
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
Enables the stable inheritance of new genes with altered expression patterns and functional domains, overcoming the limitations of existing gene editing technologies by generating new genes with improved traits and resistance/tolerance characteristics.
Implementation Method 1
simultaneously generating two or more DNA double-strand breaks at a combination of specific sites in the organism's genome
Implementation Method 2
the double-strand breaks (DSB) are repaired through the cell's own non-homologous end repair or homologous recombination mechanisms
Implementation Method 3
the double-strand breaks (DSB) are repaired through the cell's own non-homologous end repair or homologous recombination mechanisms
Data Source
AI summary
The present invention relates to the technical fields of genetic engineering and bioinformatics, in particular, to a method for creating a new gene in an organism in the absence of an artificial DNA template, and a use thereof. The method comprises simultaneously generating DNA breaks at two or more different specific sites in the organism's genome, wherein the specific sites are genomic sites capable of separating different gene elements or different protein domains, and the DNA breaks are ligated to each other through non-homologous end joining (NHEJ) or homologous repair to generate a new combination of the different gene elements or different protein domains that is different from the original genome sequence, thereby creating a new gene. The new gene of the invention can change the growth, development, resistance, yield and other traits of the organism, and has great value in application.