gRNA Array for High-Throughput TAG to TAA Genome Conversion
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Solution Overview
Problem
Current methods lack the capability for high-throughput TAG to TAA conversion in individual cells, requiring multiple sgRNAs and CBEs for efficient genome editing, which is not feasible with existing tools.
Innovation Solution
A gRNA array comprising 5 sgRNA expression cassettes connected in series, with a promoter and poly T in the 5' to 3' direction, and a base editing system including a cytosine base editor, is used for high-throughput TAG to TAA conversion, enabling efficient genome editing by co-transfecting the gRNA array with a plasmid and base editor into cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple sgRNAs and CBEs are used for efficient genome editing, then the editing efficiency is improved, but the complexity of the system increases and existing tools are not feasible
Solution Approach 1:
Multiple individual sgRNA expression cassettes are merged into a single gRNA array transcript, which is then processed by a ribozyme system to release individual sgRNAs. This combining approach maintains the functionality of multiple sgRNAs while simplifying the delivery system to a single transcript unit.
Solution Approach 2:
A ribozyme system acts as an intermediary between the gRNA array transcript and individual sgRNAs. The ribozyme sequences enable self-cleavage of the transcript to release functional sgRNAs, eliminating the need for external processing enzymes and simplifying the overall system.
2Productivity
If tens or hundreds of TAG to TAA conversions are required in a single cell, then the throughput is improved, but no tools are currently available for this application
Solution Approach 1:
The gRNA array system provides a universal platform that can accommodate tens or hundreds of different sgRNA targets within a single transcript. The modular design with standardized ribozyme sequences allows flexible configuration for high-throughput conversion applications.
Solution Approach 2:
The gRNA array transcript is segmented into multiple functional units by ribozyme sequences, allowing individual sgRNAs to be released and function independently. This segmentation enables the system to handle multiple conversion targets simultaneously while maintaining individual functionality.
3Adaptability or versatility
If individual gRNA expression cassettes are cloned into a single plasmid by Golden gate assembly, then the multiplexing capability is improved, but the device complexity and manufacturing difficulty increase
Solution Approach 1:
The ribozyme sequences enable the gRNA array transcript to self-process and release individual sgRNAs without requiring external enzymes or complex assembly procedures. This self-service mechanism eliminates the need for Golden gate assembly and other complex manufacturing steps.
Solution Approach 2:
The ribozyme system extracts individual sgRNAs from the gRNA array transcript through self-cleavage, eliminating the need for complex plasmid assembly methods. This extraction approach simplifies manufacturing while maintaining multiplexing capability.
Data Source
AI summary
A method for high-throughput TAG to TAA conversion on the genome are provided. The method comprises the following steps: co-transfecting a gRNA array pool or a transcription product thereof, a plasmid containing an mCherry-inactivated eGFP reporter molecule and an sgRNA plasmid for editing and activating eGFP in a stable cell of an inducible base editor; or by transfecting an 43-all-in-one expression vector or a transcription product thereof to cells with stable inducible base editor, high-flux TAG to TAA conversion in single cells is realized, and through multiple cyclic operations, almost all TAG to TAA conversion in the whole genome of cells of common model organisms can be realized.