off-target analysis-Gene Editing Service

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off-target analysis

Definition

Off-target effects occur when gene editing tools (such as CRISPR-Cas9) accidentally identify and cleave other similar, but not identical, sequences in the genome while targeting and cleaving a specific target DNA sequence. These unintended cleavages can lead to gene mutations, cellular dysfunction, and even cancer, and are one of the most significant safety concerns in gene therapy and clinical applications.

Working Principle and Analysis Process

The core principle of off-target effect analysis is to comprehensively screen the edited genome for unintended damage through a combination of bioinformatics prediction and experimental verification.

The standard workflow typically includes four steps:

Prediction

Using bioinformatics software, potential off-target sites that may bind to and cause cleavage are predicted genome-wide based on the gRNA (guide RNA) sequence. These sites are typically highly similar to the target sequence

Design

Specific primers are designed to target these predicted high-risk off-target sites for subsequent deep sequencing.

Detection

Whole-genome sequencing (WGS) or more economical, targeted high-throughput sequencing methods (such as AMP and GUIDE-seq) are performed on gene-edited cell samples to capture the full range of DNA changes

Validation and Analysis

Sequencing data is compared with the genome of an unedited control group to precisely locate and count the frequency and type of off-target mutations and assess their risk level.

Application Scenarios

Off-target effect analysis is crucial and primarily focuses on:

1. Basic Research: Ensuring the accuracy of gene function research results to avoid misinterpreting phenotypes caused by off-target effects as the function of the target gene.

2. Drug Development and Cell Therapy: In the development of engineered cell therapies such as CAR-T, it is imperative to ensure the accuracy of editing to prevent off-target mutations from causing cell cancer or dysfunction.

3. Clinical Gene Therapy: This is the bottom line of safety. Any clinical trial based on gene editing must provide rigorous off-target analysis data to maximize patient safety. 4. Agricultural Breeding: When developing genetically modified crops, off-target effects must be eliminated to ensure genetic stability and environmental safety.

Advantages and Importance

Improving Safety

Directly identifying and assessing potential risks is the cornerstone for advancing gene editing technology toward clinical application.

Ensuring Data Reliability

In scientific research, it can effectively avoid false positives in experimental results, enhancing the rigor and reproducibility of research.

Guiding Tool Optimization

Analysis results can be used to inform researchers and design more specific and high-fidelity gene editing tools (such as high-fidelity Cas9 variants), thereby reducing off-target effects at the source.

In summary, off-target effect analysis is not simply an additional experiment; it is an essential component of responsible gene editing. Like a precise "genome quality inspector," it provides the most critical technical guarantee for the safe and effective application of this powerful technology, safeguarding the future of gene editing.

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Definition

Improving Safety

Ensuring Data Reliability

Guiding Tool Optimization

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