Prime Editing Tag Insertion for Accurate Off-Target Prediction
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Solution Overview
Problem
Existing methods for predicting off-targets in genome editing are not suitable for prime editing systems, as they are developed for traditional CRISPR/Cas systems, leading to challenges in accurately identifying and mitigating side effects in prime editing processes.
Innovation Solution
A method for predicting off-targets in prime editing systems involves obtaining manipulated genome DNA with a tag sequence inserted by a prime editor protein and tpegRNA, analyzing the tagmentation to identify off-target candidates, and verifying on-target information for precise off-target prediction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional CRISPR/Cas system prediction methods are used, then the prediction process is simple, but the accuracy is insufficient for prime editing systems
Solution Approach 1:
The method performs preliminary actions by inserting a tag sequence into the genome DNA before off-target analysis. This tag insertion creates a detectable marker that enables subsequent identification of off-target sites through tag-specific sequencing, thereby improving prediction accuracy without requiring complex external tools
Solution Approach 2:
The tag sequence acts as an intermediary element between the prime editing system and the detection method. By incorporating this intermediate tag into the genome DNA at potential off-target sites, the system enables indirect detection through sequencing, resolving the contradiction between simplicity and accuracy
2Measurement precision
If tag sequence insertion is performed for off-target prediction, then off-target identification accuracy is improved, but the editing process complexity increases
Solution Approach 1:
The method merges the off-target prediction function with the prime editing process itself. The tag insertion step is integrated into the editing workflow, and the same sequencing technology used for editing verification is leveraged for off-target detection, thereby improving accuracy without proportionally increasing overall process complexity
Solution Approach 2:
The tag sequence serves multiple functions: it acts as a marker for off-target detection, a template for reverse transcription, and a verification element for editing accuracy. This multi-functionality reduces the need for separate detection systems, balancing improved identification accuracy with manageable process complexity
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
The method provides accurate identification of off-target regions and candidates in prime editing, enhancing the safety and precision of genome editing by leveraging the molecular mechanisms of prime editing systems.
Implementation Method 1
the tag sequence is inserted into the genome DNA by reverse transcription process performed by the reverse transcriptase using the tag template of the tpegRNA as a template of reverse transcription
Data Source
AI summary
The present application relates to a method for predicting an off-target which can occur in the process of editing a genome by using a prime editing system.