TA-AS Method for Detecting Base Editing Off-Targets
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Solution Overview
Problem
Current methods for detecting genome-wide random off-target effects of base editing systems are inefficient and cannot be performed in a high-throughput mode, limiting their application in genetic engineering.
Innovation Solution
The method involves co-transferring a base editing system with an orthogonal CRISPR system that generates a stable single-stranded DNA region, allowing for efficient detection of off-target effects through amplicon high-throughput sequencing, known as Trans-ssDNA amplicon deep sequencing (TA-AS) method.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If genome-wide sequencing technology is used to evaluate off-target effects, then detection accuracy is improved, but cost and time efficiency deteriorate
Solution Approach 1:
The invention segments the genome-wide sequencing task into targeted amplicon sequencing of specific regions. Instead of sequencing the entire genome, the method focuses on amplifying and sequencing only the regions containing single-stranded DNA structures, which are the potential off-target sites. This segmentation maintains detection accuracy for relevant regions while dramatically reducing cost and time requirements.
Solution Approach 2:
The invention extracts the critical detection target (single-stranded DNA regions) from the complex genome-wide sequencing process. By using enrichment strategies to isolate and amplify only the regions with single-stranded DNA structures, the method removes unnecessary sequencing of irrelevant genomic regions, achieving high-throughput detection without sacrificing accuracy for the regions of interest.
2Measurement precision
If genome-wide sequencing is performed to detect random off-target effects, then detection coverage is improved, but processing time increases
Solution Approach 1:
The invention performs preliminary enrichment of single-stranded DNA regions before sequencing. By using molecular biology techniques to pre-isolate and amplify the regions containing single-stranded DNA structures, the method prepares the sample in advance, ensuring that only relevant regions are subjected to sequencing. This preliminary action reduces processing time while maintaining comprehensive detection coverage of potential off-target sites.
3Productivity
If high-throughput detection method is implemented, then productivity is improved, but detection sensitivity may deteriorate
Solution Approach 1:
The invention introduces amplicon sequencing as an intermediary step between genome-wide sequencing and off-target detection. This intermediary approach uses targeted amplification and sequencing of specific regions, which maintains high sensitivity for detecting off-target effects while enabling high-throughput processing. The intermediary method acts as a bridge that preserves detection sensitivity without requiring full genome-wide sequencing.
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 enables rapid and cost-effective detection of random off-target effects, correlating well with genome-wide sequencing results and providing high sensitivity and accuracy for evaluating base editing systems.
Implementation Method 1
a CRISPR detection system which targets at least one detection target site in the genome into the cell or the organism, wherein the CRISPR detection system being capable of forming a single-stranded DNA region at the at least one detection target site
Implementation Method 2
the base editor which can randomly act on a single-stranded DNA region deaminates on the target bases of the single-stranded region
Implementation Method 3
the random off-target effect of the base editing system can be efficiently, simply and conveniently detected by amplicon high-throughput sequencing
Data Source
AI summary
The present invention belongs to the field of gene editing, and particularly relates to a method and means for detecting genome wide random off-target effect of a base editing system in a rapid and high-throughput mode.