Balanced Indels for Precise DNA Modification
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Solution Overview
Problem
Current methods for modifying genetic material in eukaryotic organisms lack precision, as they do not allow for controlled insertion of exogenous DNA fragments into specific genomic locations, leading to unpredictable expression levels and potential disruption of balanced genomes.
Innovation Solution
The use of two site-specific nucleases to introduce targeted alterations in a coding sequence of duplex DNA, allowing for precise modification of specific parts of a protein without disrupting the reading frame, thereby enabling the alteration of predetermined amino acid stretches.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional methods of adding exogenous DNA fragments to the genome are used, then new properties can be conferred to cells, but the insertion positions are not precisely controlled and expression levels are unpredictable
Solution Approach 1:
The genome editing process is segmented into distinct functional components: guide RNAs that recognize specific target sequences, nucleases that create controlled breaks at those sequences, and repair mechanisms that introduce precise modifications. This segmentation allows independent optimization of each component for both versatility and precision.
Solution Approach 2:
Guide RNAs serve as intermediaries that bridge the gap between the desired target sequence and the nuclease enzyme. The guide RNA contains a spacer sequence complementary to the target, acting as a molecular address label that directs the nuclease to the exact genomic location, thereby achieving precise control over insertion positions while maintaining versatility through programmable guide RNA design.
2Manufacturing precision
If genome editing methods are used to add or delete nucleotides in predefined loci, then precise modification of existing genes is achieved, but the reading frame may be disrupted
Solution Approach 1:
The invention changes the parameter of indel size to be a multiple of three nucleotides (3, 6, 9, etc.). This parameter change ensures that while the coding sequence is modified precisely at the target location, the reading frame is preserved because three-nucleotide increments correspond to complete amino acid codons, thereby maintaining protein integrity while achieving precise gene modification.
Solution Approach 2:
Instead of making minimal single-nucleotide changes that might disrupt the reading frame, the method applies a controlled excessive action by introducing larger indels that are specifically sized (multiples of three) to maintain the reading frame. This partial action approach modifies only the necessary portion of the gene while preserving the overall structure and reading frame.
3Manufacturing precision
If single indels are introduced in coding sequences, then nucleotide changes are achieved, but the reading frame is shifted and protein function is lost
Solution Approach 1:
The invention applies a counterweight principle by introducing a second indel that compensates for the reading frame disruption caused by the first indel. The two indels are positioned and sized such that their combined effect restores the reading frame, with the second indel acting as a corrective element that counterbalances the frame-shifting effect of the first indel, thereby preserving protein function while achieving the desired nucleotide changes.
Solution Approach 2:
The genetic modification strategy uses a composite approach combining multiple indel events (first indel and second indel) to achieve the desired outcome. Just as composite materials combine different components to achieve properties that individual components cannot provide alone, this method combines multiple indel events to simultaneously achieve precise nucleotide changes while maintaining the reading frame and protein function.
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 method enables the precise modification of proteins by altering specific parts of the coding sequence, allowing for the creation of novel or improved protein functions, such as enhanced herbicide resistance, while maintaining the overall reading frame and protein stability.
Implementation Method 1
a first site-specific nuclease cleaves the DNA generating a first indel at a first location within the ORF and wherein a second site-specific nuclease cleaves the DNA generating a second indel at a second location within the same CDS
Data Source
AI summary
The invention pertains to the targeted alteration of a duplex DNA in a cell, whereby two site-specific nucleases generate an indel, such that the open reading frame is not altered after the second indel. The invention further pertains to the use of such nucleases for the targeted alteration of an open reading frame in duplex DNA and a kit of parts for use in a method of the invention. Using the method of the invention, novel plants were obtained having an improved herbicide resistance. The invention therefore also concerns plants having improved herbicide resistance due to the expression of an altered ALS protein.