Genome Editing Selectable Marker Ratios and Digital PCR Quantification
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Solution Overview
Problem
Current genome editing methods face challenges in achieving high specificity and efficiency, particularly due to off-target effects and random integration, when modifying target sites in eukaryotic cells, and lack quantitative methods for evaluating knock-in efficiency.
Innovation Solution
A method involving the introduction of specific nucleic acids encoding an RNA-guided nuclease, guide RNA, and a selectable marker, with controlled ratios, followed by digital PCR using probe pairs to detect and quantify the presence of exogenous nucleic acid sequences at predetermined loci, allowing for precise selection and evaluation of modified cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a site-specific nuclease is used to introduce DNA double-strand breaks for genome editing, then gene insertion via homologous recombination can be achieved, but off-target effects and random integration occur reducing specificity
Solution Approach 1:
The patent introduces a selectable marker gene along with the homology arm-containing nucleic acid sequence before performing homologous recombination. This preliminary action enables subsequent selection of cells that have successfully undergone targeted integration, thereby distinguishing them from cells with random integration or off-target effects, and improving genome editing specificity
Solution Approach 2:
The patent employs a selection system where cells expressing the selectable marker (indicating successful targeted integration) can be identified and isolated. This feedback mechanism allows researchers to screen and select only those cells that have achieved the desired genome modification, eliminating cells with off-target effects or random integration events
2Measurement precision
If conventional PCR is used to detect amplification products for confirming gene insertion, then presence of exogenous nucleic acid can be detected, but quantitativeness is lacking preventing evaluation of knock-in efficiency
Solution Approach 1:
The patent replaces conventional qualitative PCR detection with digital PCR technology. Digital PCR divides the sample into numerous individual reactions, allowing absolute quantification of target nucleic acid sequences. This substitution enables precise measurement of knock-in efficiency by quantifying the ratio of edited to unedited alleles, providing the missing quantitativeness
3Manufacturing precision
If a targeting vector with homology arms is used for knock-in through homologous recombination, then gene insertion can be achieved, but the process is complex and time-consuming
Solution Approach 1:
The patent segments the traditional targeting vector into separate components: a nucleic acid sequence containing only the essential homology arms and selectable marker, excluding unnecessary elements. This segmentation simplifies the construction process while maintaining the ability to achieve precise gene insertion through homologous recombination
4Object-affected harmful factors
If truncated guide RNA is used to increase specificity of genome editing, then off-target effects are reduced, but guide RNA design becomes more constrained
Solution Approach 1:
The patent changes the parameter of guide RNA length to optimize the balance between specificity and design flexibility. By using truncated guide RNAs with reduced complementarity regions, the patent achieves lower off-target effects while the modular design approach maintains adaptability for different target sites through systematic optimization of the truncated sequences
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 enhances the specificity and efficiency of genome modifications by reducing off-target effects and random integration, while providing a quantitative method to assess knock-in efficiency, thereby improving the accuracy of genetic modifications in eukaryotic cells.
Implementation Method 1
The gRNA comprises a complementary region that binds to a DNA sequence of the target site through base-pairing
Implementation Method 2
a method involving performing digital PCR on the genome as a template
Implementation Method 3
detecting, via a digital PCR method, the presence or absence of hybridization, to a genomic DNA fragment derived from the chromosome, of a first probe nucleic acid that hybridizes to all or part of the nucleic acid sequence to be detected
Data Source
AI summary
Provided is a method of modifying a target site in the genome of a eukaryotic cell, the method comprising: (1) a step of introducing into the cell, introduction nucleic acids comprising (a) a template nucleic acid comprising a nucleic acid sequence encoding an RNA-guided nuclease, (b) a template nucleic acid comprising a nucleic acid sequence encoding a guide RNA, or a guide RNA, and (c) a template nucleic acid comprising a nucleic acid sequence encoding a selectable marker; and (2) a step of selecting a cell expressing the selectable marker, wherein the number of moles (C) of (c) the template nucleic acid comprising a nucleic acid sequence encoding a selectable marker, subjected to the step (1), is smaller than any of the number of moles (A) of (a) the template nucleic acid comprising a nucleic acid sequence encoding an RNA-guided nuclease and the number of moles (B) of (b) the template nucleic acid comprising a nucleic acid sequence encoding a guide RNA, or the guide RNA.