Target DNA Fragment Duplication Using Complementary pegRNA Templates
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Solution Overview
Problem
Current CRISPR-based technologies, such as prime editing, are inefficient in duplicating larger DNA fragments and require new RT templates for each target portion, limiting their application in genetic disease treatment and industrial protein production.
Innovation Solution
Amplification Editing (AE) method using a pair of pegRNA with complementary RT templates to extend and duplicate target DNA fragments, forming a duplex region for DNA polymerase to synthesize new strands, thereby amplifying the target fragment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If prime editing is used to insert DNA fragments, then precise modification can be achieved, but the length of duplicated fragment is limited and new RT templates are required for each target portion
Solution Approach 1:
The invention divides the RT template into multiple segments distributed across different pegRNAs. Each pegRNA carries a portion of the template sequence, and through sequential reverse transcription events, the complete template is reconstructed to enable duplication of large DNA fragments without requiring a single long RT template
Solution Approach 2:
The invention creates a universal amplification system where a standardized set of components (Cas protein, DNA polymerase, and modular pegRNAs) can duplicate any target fragment within the achievable length range. The system replaces the need for custom-designed long RT templates with a flexible combination of shorter, interchangeable pegRNA modules
2Manufacturing precision
If homology-directed repair (HDR) is used for targeted transgene integration, then precise integration can be achieved, but efficiency is low in non-dividing cells and limited by exogenous DNA donor
Solution Approach 1:
The invention replaces the mechanical process of exogenous DNA donor delivery and homology search (HDR mechanism) with a biochemical template-directed synthesis mechanism. The RT template provides direct sequence information for duplication, eliminating the need for exogenous DNA donors and making the process independent of cell division cycles
Solution Approach 2:
The system uses the target DNA sequence itself as the template for duplication. The RT template is designed to anneal to the target sequence, and the reverse transcription and DNA polymerase activities use the target DNA as the basis for generating duplicate copies, eliminating dependence on external donor DNA
3Productivity
If HITI strategy is used for targeted integration, then cell cycle independence can be achieved, but efficiency remains low at genomic level with mixed integration events
Solution Approach 1:
The invention introduces an RNA-based intermediary (RT template within pegRNA) that mediates the duplication process. This RNA template provides precise sequence information that guides the duplication reaction, ensuring high specificity at the intended target site and preventing off-target or mixed integration events while maintaining cell cycle independence
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
AE achieves efficient and precise duplication of target fragments, ranging from 20 bp to 100 Mb, with high accuracy and robustness across various cell lines and genomic loci, including recurring amplification without disrupting flanking sequences.
Implementation Method 1
the reverse transcriptase to extend the two opposite strands of the target fragment, with the first and second RT template sequences as templates to generate two single-stranded flap DNA sequences
Implementation Method 2
As the two RT templates at least include portions that are complementary to each other, they can form a duplex region which can then serve as starting point for DNA polymerase to synthesize a new strand
Implementation Method 3
the DNA polymerase to extend the double-stranded region to duplicate each strand of the target fragment, thereby duplicating the target fragment
Data Source
AI summary
Provided are compositions and methods useful for duplicating/amplifying a target fragment on a target DNA sequence such as a genome sequence. The editing system employs a pair of pegRNA which, by virtue of their targeting sites flanking the target fragment, extend the target fragment with reverse transcriptase (RT) templates included in the pegRNA. As the two RT templates at least include portions that are complementary to each other, they can form a duplex region which can then serve as starting point for DNA polymerase to synthesize a new strand for each strand of the target fragment, thereby duplicating the target fragment. Continue this process introduce amplification of this targeted sequence. Alternatively, this process can be done by combination of pegRNA/sgRNA or sgRNA/sgRNA. In the case of sgRNA/sgRNA in a PAM-out position, the RT enzyme and templates are not required.