Liquid-Phase Capture Off-Target Verification

Liquid-Phase Capture Off-Target Verification

1. Background

According to the latest FDA guidance document on gene therapy products (January 2024), regulatory agencies have put forward comprehensive requirements for the safety evaluation of gene therapy products incorporating human genome editing. The guidance particularly emphasizes the importance of identification and verification of off-target editing events, including full analysis of type, frequency, and location. The regulatory requirement states: "Verification of off-target sites should be conducted using methods with adequate sensitivity to detect low frequency events" and that off-target sites identified through three approaches — software prediction, GUIDE-seq cell-based detection, and AID-seq in vitro detection — must all be subject to verification. Furthermore, the verification must be conducted in at least three independent donor samples and accompanied by unedited controls to filter background variants.

Figure 1. FDA guidance document Human Gene Therapy Products Incorporating Human Genome Editing: requirements for off-target site verification

Under this regulatory landscape, liquid-phase hybrid capture off-target verification has emerged as a genome-wide unbiased off-target detection method. Unlike amplicon-based off-target verification, which requires individually designing primers and constructing libraries for each potential off-target site, liquid-phase hybrid capture technology uses a probe panel covering all off-target genomic regions to simultaneously capture hundreds to thousands of potential off-target sites in a single reaction, substantially improving detection efficiency and comprehensiveness. It is generally used as a preliminary screening tool for efficient high-frequency off-target site identification (editing efficiency ≥0.2%), meeting regulatory requirements.

2. Liquid-Phase Hybrid Capture Off-Target Verification Principle

The fundamental principle of Targeted Enrichment Sequencing (TES) is based on complementary base pairing: a set of biotinylated probes covering the target sequences is designed to collect and detect specific DNA fragments. When a target sequence is present in the sample genome, it hybridizes specifically with the probes. The extracted genomic DNA fragments are hybridized with the probes; using streptavidin and magnetic beads, DNA fragments containing the target sequences can be specifically enriched. Liquid-phase hybrid capture technology has been applied to the off-target safety evaluation of numerous gene-editing therapy products including CASGEVY, and is the most widely adopted genome-wide off-target detection method in the industry, providing reliable technical support for quality control of gene-editing products.

The TES detection workflow primarily comprises the following steps: first, library construction is performed on the DNA sample to be tested — genomic DNA is randomly fragmented by sonication to 150–300 bp; following end repair and dA-tailing, specific adapters containing UMI sequences are ligated, assigning a unique molecular barcode to each DNA molecule. Subsequently, based on target sequence information, specific probes are synthesized; the UMI-barcoded library is hybridized with the probes and subjected to dual capture using streptavidin and magnetic beads to enrich DNA fragments containing target sequences. Finally, target signal-enriched sequencing libraries are amplified by PCR and constructed; at the bioinformatics analysis stage, UMI sequences are used for molecular-level deduplication to achieve precise quantification of target sequences. Compared with conventional methods, the UMI-based liquid-phase hybrid capture approach not only more comprehensively captures target sequences, but also eliminates PCR amplification bias through molecular barcode technology, significantly improving the accuracy, sensitivity, and quantification precision of detection.

Figure 2. Schematic illustration of the liquid-phase hybrid capture sequencing principle

3. Detection and Analytical Advantages of Liquid-Phase Hybrid Capture Off-Target Verification

3.1 Highly Efficient Platform for Multi-Site Simultaneous Verification

(1) Multi-site parallel analysis: a single experiment can simultaneously capture and analyze hundreds to thousands of potential off-target sites, substantially increasing detection throughput,   compared with amplicon-based methods that require individually designing primers and constructing libraries for each site, efficiency is improved 10–100-fold;

(2) Standardized workflow: all sites are captured and sequenced under identical experimental conditions, eliminating batch effects from multiple library constructions and sequencing runs, and improving data comparability;

(3) Resource utilization optimization: capturing multiple sites in a single run significantly reduces reagent consumption and labor costs while reducing sequencing resource waste, particularly suitable for large-scale off-target safety evaluation screening, only one round of DNA extraction and library preparation is required, greatly reducing sample consumption — especially advantageous for precious sample analysis.

3.2 Outstanding Data Quality and Accuracy

(1) UMI technology reduces false-positive rate: by integrating a unique molecular identifier (UMI) technology in sequencing adapters, each original DNA molecule is assigned a unique molecular barcode; bioinformatic analysis can effectively identify and eliminate errors introduced during PCR amplification and sequencing, reducing the false-positive rate from ~1% for conventional methods to below 0.01%, ensuring data authenticity;

(2) High-sensitivity detection: benefiting from UMI technology and an optimized experimental workflow, liquid-phase hybrid capture can reliably detect down to 0.2%, accurately identifying individual cells carrying off-target editing events among 500 normal cells, providing strong technical support for low-frequency off-target event detection.

3.3 Service Advantages

(1) Leading technology platform: capable of comprehensive detection of multiple off-target mutation types induced by gene editing, including substitutions, insertions, and deletions, meeting safety evaluation needs at all stages of biological research;

(2) Scientifically rigorous off-target site filtering strategy;

(3) Comprehensive cancer-associated gene annotation: detailed tumor gene annotation for every high-frequency (≥0.2%) off-target site; ONCOGENE database information integrated to annotate cancer-associated oncogenes, combined with TSG (tumor suppressor gene) data to identify tumor suppressor genes, providing complete gene functional descriptions and potential risk assessments;

(4) Rich success case portfolio: services have been successfully delivered to numerous domestic and international gene therapy companies, supporting smooth IND submissions for their products.

4. Case Studies

The liquid-phase hybrid capture off-target verification analytical reports provided by the ZhuHai GeneRulor adopt a standardized format to ensure professional and comprehensive data presentation. The introductory section covers project background, experimental rationale, technical principles, library construction workflow description, and bioinformatics analysis pipeline overview, providing clients with the necessary technical context. This is followed by sample information and sequencing data statistics, which include sample metadata, raw sequencing data quality metrics, and control group alignment result statistics, ensuring data quality and reliability. In addition, the report includes the following key components:

(1) Provision of a scientifically rigorous off-target site filtering strategy, showing the number of sites retained at each filtering step and the final number of retained off-target sites.

(2) Provision of positional information for all high-frequency (≥0.2%) off-target sites, along with raw mutation rates and background-filtered mutation rates, to facilitate assessment of the true off-target editing level.

Figure 3. Representative display of detailed information for high-frequency (≥0.2%) off-target sites per sample (illustrative example)

(3) Not only focusing on the frequency of off-target sites, but also conducting in-depth analysis of their potential biological significance — particularly their relevance to tumor onset and progression. By distinguishing among ordinary genes, tumor suppressor genes (TSGs), and oncogenes, off-target sites are risk-stratified to prioritize attention to high-risk sites.

Figure 4. Functional annotation of high-frequency (≥0.2%) off-target sites per sample (illustrative example)

(4) Provision of visualization analysis for high-frequency off-target sites: precise alignment between reference sequence and edited sequence; clear marking of sgRNA binding sites and potential cleavage sites; direct visualization of editing types. Visualization results simultaneously display the read count and mutation rate of both the experimental group and the control group, filtering background noise by subtracting control group background editing efficiency from experimental group editing efficiency. Sites with background-filtered editing efficiency ≥0.2% are identified as high-frequency off-target sites. This molecular-level precise analysis enables decision-makers to directly understand the specific changes at editing sites and to distinguish genuine gene-editing signals from sequencing background noise.

Figure 5. Mutation result visualization for high-frequency off-target sites (≥0.2%) (illustrative example)

6. Service Content

* Service turnaround: standard workflow 20–30 business days.

7. Sample Requirements

* Notes: (1) All samples must meet the above standards to ensure the accuracy and reliability of detection results. (2) Clients may also submit tissue or cell pellet samples for DNA extraction; tissue requirement: >50 mg; cell pellet requirement: >1×10⁵ cells per site. (3) For special sample types, please contact the ZhuHai GeneRulor technical team in advance (Tel: 400-6309596; Order/Technical Support: service@generulor.com).

8. References

[1] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). (2005). ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology Q2(R1) [current 4th edition].

[2] U.S. Food and Drug Administration. (May 2018). Bioanalytical Method Validation: Guidance for Industry. U.S. Department of Health and Human Services, FDA Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM).

[3] FDA. Human Gene Therapy Products Incorporating Human Genome Editing – Guidance for Industry. 2024.