Double-pegRNA Prime Editing for Precise Plant Genome Insertion
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Solution Overview
Problem
Current genome editing tools, particularly the CRISPR/Cas system, face challenges in achieving efficient and precise site-directed insertion of large DNA fragments into plant genomes, due to low frequency of repair pathways and instability of reverse transcription components.
Innovation Solution
The use of a double-pegRNA strategy within the prime editing system (PE), coupled with enhanced reverse transcription capabilities through ePPE and epegRNA, and the integration of site-specific recombinase systems like Cre/Lox or FLP/FRT, to facilitate efficient and precise insertion of exogenous sequences into plant genomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the CRISPR/Cas system is used for site-directed insertion of exogenous sequences, then the precision of insertion is improved, but the efficiency of insertion is worsened due to low frequency of repair pathways
Solution Approach 1:
The patent combines the CRISPR/Cas system with homologous recombination templates and repair pathway enhancers into an integrated editing system. This merging enables simultaneous activation of precise insertion through CRISPR-guided DNA break formation and efficient repair through enhanced homologous recombination, resolving the contradiction between precision and efficiency
Solution Approach 2:
The patent modifies key parameters of the CRISPR/Cas system including PAM sequence requirements, guide RNA design, and repair template characteristics to optimize both precision and efficiency. By changing these parameters, the system achieves high-frequency precise insertions that overcome the limitations of traditional CRISPR/Cas alone
2Measurement precision
If the prime editing system is used for insertion of exogenous sequences, then the precision is improved, but the stability of reverse transcription components is worsened
Solution Approach 1:
The patent incorporates stabilizing elements and protective sequences into the reverse transcription template design before the editing process occurs. These pre-built stabilizing components protect the reverse transcription machinery from degradation and maintain structural integrity throughout the editing process, ensuring both precision and stability
Solution Approach 2:
The patent uses composite reverse transcription templates combining multiple functional elements (homologous arms, selection markers, stabilizing sequences) to create a robust editing component. This composite structure provides both the precision needed for site-directed insertion and the stability required for successful reverse transcription
3Reliability
If traditional crop breeding methods are used, then the reliability of trait inheritance is improved, but the time required and labor intensity are worsened
Solution Approach 1:
The patent replaces the mechanical and time-consuming traditional breeding processes (crossing, selfing, selection) with a molecular biology-based genome editing system. This substitution enables direct modification of target genes without requiring multiple generations of crossing and selection, dramatically reducing time and labor while maintaining inheritance reliability
Solution Approach 2:
The patent performs genetic modifications at the molecular level before traditional breeding operations would be required. By pre-editing the genome with desired traits through CRISPR/Cas or prime editing, the system eliminates the need for lengthy breeding cycles, achieving both speed and reliability
4Productivity
If transgenic technology is used for introducing foreign genes, then the efficiency of obtaining excellent traits is improved, but the regulatory complexity is worsened
Solution Approach 1:
The patent extracts and eliminates foreign gene elements from the editing system, achieving trait improvement through precise modification of endogenous genes. By taking out the need for foreign transgenes and using only genome editing tools, the system maintains high efficiency for obtaining excellent traits while significantly reducing regulatory complexity
Solution Approach 2:
The patent uses transient, non-heritable editing components (CRISPR/Cas proteins, guide RNAs) that disappear after performing their editing function, replacing permanent foreign gene integration. This approach achieves efficient trait acquisition without the long-term regulatory burdens associated with transgenic organisms
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 significantly enhances the efficiency of site-specific insertion, achieving precise integration of large DNA fragments with improved stability and accuracy, thereby overcoming the limitations of existing genome editing technologies in plant cells.
Implementation Method 1
a reverse transcriptase and/or an expression construct comprising a nucleotide sequence encoding the reverse transcriptase
Implementation Method 2
sgRNA targets genomic DNA at a specific location through the principle of complementary base pairing
Implementation Method 3
the RuvC and HNH nuclease active domains on it are activated to complete the cleavage of the non-target strand and the target strand respectively to generate a DNA double-strand break (DSB)
Implementation Method 4
the endogenous repair mechanism of the cell may use the donor as a template to perform homologous recombination repair (HR)
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3
AI summary
The invention belongs to the field of genetic engineering. Specifically, the present invention relates to a method for site-directed insertion of exogenous sequence in a genome. Specifically, based on the prime editing system (PE), the present invention uses two adjacent pegRNAs with partially overlapping sequences on the reverse transcription template to achieve efficient and precise site-directed insertion of exogenous sequences in the genome, especially genome of a plant. The system is further coupled with a recombinase system such as Cre/Lox or FLP/FRT, etc., to achieve site-directed insertion of large fragment of exogenous sequences in the genome, especially genome of a plant.