TGS PEM-seq HR Efficiency Detection

TGS PEM-seq HR Efficiency Detection

1. Background

Gene-editing technology plays an important role in gene therapy, disease model construction, and functional genomics research. Among the available approaches, homology arm-mediated gene knock-in strategy based on Homologous Recombination (HR) represents a critical tool for achieving precise gene editing. This strategy involves designing a donor template (Donor) that carries the homologous sequences; following the generation of a DNA double-strand break (DSB) by gene-editing tools such as CRISPR-Cas9, the cell's own homologous recombination repair machinery is leveraged to precisely integrate the target gene at the desired locus.

However, HR-mediated gene integration typically involves the insertion of relatively long DNA sequences (commonly ranging from hundreds of base pairs to several kilobases), posing significant technical challenges for detection. Conventional NGS-based second-generation PEM-seq approaches are constrained by the 150 bp read length limitation of PE150 sequencing, making it impossible to span large insertion regions and thus unable to accurately detect integration events or assess integration efficiency. Furthermore, for gene-editing projects involving HR-mediated integration, it is also necessary to clearly distinguish among complete integration, partial integration, random insertion, and other different repair outcomes — requirements that demand detection methods with sufficient read length and high accuracy.

To address these technical bottlenecks, ZhuHai GeneRulor has introduced a third-generation PEM-seq detection technology based on the PacBio long-read sequencing platform. By overcoming the read length limitation of second-generation sequencing, this approach achieves full-length coverage of long DNA insertions, enables precise detection of HR integration events, comprehensively evaluates gene-editing integration efficiency and accuracy, and provides a key quality control tool for the research and clinical translation of gene therapy products.

Figure 1. HR-mediated gene integration strategy and the limitations of second-generation sequencing technology

2. Detection Principle of Third-Generation PEM-seq

Third-generation PEM-seq is an upgraded version developed on the basis of second-generation PEM-seq. Its core principle remains the use of specific bait primers (lure primers) designed in the vicinity of the DSB site to capture sequences that rejoin at the DSB locus (prey sequences), but employs the PacBio long-read sequencing platform for long-read-length sequencing, thereby achieving comprehensive detection of long DNA insertions. The key workflow steps are as follows:

(1) Sample DNA is prepared and subjected to tagmentation processing to reduce PCR bias through UMI molecular barcoding, ensuring accurate quantification. Subsequently, site-specific bait primers are used to enrich toward the target region, expanding the coverage of target genomic sequences, homology arm sequences, and partial insertion sequences.

(2) Following enrichment, products are purified using streptavidin-coated magnetic beads; a sequencing library is then constructed and loaded onto the PacBio HiFi sequencer to obtain long reads of high accuracy, enabling full-length coverage and precise analysis of insertion fragments.

3. Detection and Analytical Advantages of Third-Generation PEM-seq

Based on the PacBio long-read sequencing platform, third-generation PEM-seq technology enables precise detection of the two key junction regions — Genome–Homology Arm junction and Homology Arm–Insertion junction — to verify integration accuracy; it simultaneously comprehensively identifies multiple integration patterns including correct integration, reverse integration, tandem multi-copy concatenation, and truncated insertion, providing key data support for gene-editing safety evaluation.

Figure 2. Third-generation PEM-seq: precise junction detection and comprehensive integration pattern analysis

3.1 Long-Read-Length Detection

The PacBio sequencing platform provides long-read-length sequencing capability, with complete coverage of amplicon sequence length (primarily 2–3 kb), enabling precise identification of key junction regions:

(1) Genome–Homology Arm junction: verification of correct homology arm integration;

(2) Homology Arm–Insertion junction: confirmation of insertion sequence connection accuracy;

(3) Integration structure completeness: detection of the integrity of the homology arm and partial insertion sequences;

(4) Complex integration patterns: identification of complex structures such as reverse insertions and tandem multi-copy concatenations.

3.2 High-Accuracy Quantitative Analysis

Leveraging UMI molecular barcode technology, third-generation PEM-seq achieves precise quantification of different integration events:

(1) Integration efficiency assessment: accurate calculation of the proportion of successful integration events among all editing events;

(2) Integration event quantitative analysis: based on junction sequence characteristics, precise calculation of insertion event frequency; for different integration events at the same target site, discrimination and classification statistics can be performed through sequence differences within the amplification region;

(3) Background noise control: effective elimination of background interference through control group comparison.

3.3 Comprehensive Structural Variant Detection

While detecting integration events, third-generation PEM-seq retains all the detection capabilities of second-generation PEM-seq:

(1) Small indels (Indel);

(2) Large deletions (Large Deletion);

(3) Chromosomal translocations (Translocation);

(4) Chromosomal inversions (Inversion);

(5) Off-target effect assessment.

3.4 High-Quality Data Output

(1) Sequencing accuracy: PacBio HiFi mode accuracy up to 99.9%, comparable to second-generation sequencing;

(2) Coverage depth: thousands to tens of thousands of effective reads obtainable per target site;

(3) Data reliability: UMI deduplication and multi-layer quality control ensure authentic and reliable data.

4. Application Scenarios of Third-Generation PEM-seq

4.1 AAV/Lentivirus-Mediated Gene Therapy

(1) HDR-mediated gene correction;

(2) Therapeutic gene site-directed integration;

(3) Safe harbor site gene knock-in;

(4) Integration efficiency and safety assessment.

4.2 CAR-T / CAR-NK Cell Therapy

(1) CAR gene site-directed integration;

(2) TCR gene knockout combined with CAR gene knock-in;

(3) Multi-gene simultaneous editing integration detection.

4.3 Disease Model Construction

(1) Site-directed integration of disease-associated genes;

(2) Reporter gene knock-in;

(3) Conditional gene knock-in validation.

4.4 Functional Genomics Research

(1) Tagged protein fusion expression;

(2) Gene overexpression / knock-in validation;

(3) Post-CRISPR library screening integration validation.

4.5 IND Submission Support

(1) Integration efficiency testing of gene therapy products;

(2) Integration site accuracy validation;

(3) Unintended integration event assessment;

(4) Establishment of key quality control indicators.

5. Representative Third-Generation PEM-seq Detection Report

ZhuHai GeneRulor provides detailed and professional integration analysis reports that fully meet IND submission requirements for gene therapy products. The core content of the report includes:

(1) Sample Information and Sequencing Quality Assessment: The report front page includes complete sample traceability information (project number, submitting organization, sample information, date, etc.) and sequencing quality metrics (sequencing platform, data output, coverage depth, Q30, etc.). PCR deduplication is performed using UMI molecular barcode technology to ensure the accuracy of quantitative results.

(2) Junction Sequence Analysis and Integration Efficiency Assessment: Based on PacBio long-read sequencing (2–5 kb), complete coverage of the Genome–Homology Arm junction and Homology Arm–Insertion junction regions is achieved to precisely verify the accuracy of homology arm integration and the completeness of insertion sequence connection. Detailed junction region sequence information, read support data, and precise insertion event frequency calculations are provided. For different integration events at the same target site, discrimination and classification statistics are performed through sequence differences within the amplification region.

(3) Complex Integration Pattern and Structural Variant Comprehensive Detection: Multiple integration patterns are identified and quantitatively analyzed (forward/reverse integration, single-copy/multi-copy tandem concatenation, complete/truncated insertion, etc.); CRISPR editing-induced structural variant events such as large deletions (>1 kb), chromosomal translocations, and inversions are comprehensively detected to assess genome integrity and product safety.

(4) Data Visualization and Quality Control Information: IGV genome browser visualization, integration event summary tables, structural variant detection results, and more are provided. The report includes complete detection method description, quality control parameters, and data interpretation, meeting regulatory submission requirements.

For a complete sample report, please contact us.

6. Third-Generation PEM-seq Service Content

* Service turnaround: standard workflow 35–40 business days.

* Service highlights:

(1) Integrated end-to-end service available, from Donor design and sgRNA design through final data analysis;

(2) Supports single-site, dual-site, and multi-site detection;

(3) Customized analytical solutions available.

7. Sample Requirements

7.1 Basic Service Options

(1) End-to-end service: full-process service from Donor plasmid construction, cell transfection, and DNA extraction through sequencing analysis is available (client provides cell line);

(2) Library construction and sequencing service: client provides qualified DNA samples; ZhuHai GeneRulor provides library construction, sequencing, and analysis services;

(3) Sequencing and analysis service only: client provides library products; ZhuHai GeneRulor provides sequencing and analysis services.

7.2 DNA Sample Standards

7.3 Cell Sample Standards

7.4 Experimental Grouping Requirements

It is strongly recommended to submit both the experimental group (post-gene-editing) and the control group (unedited) samples simultaneously, for accurate assessment of integration efficiency and elimination of background interference.

7.5 Required Information to Be Provided by Client

It is strongly recommended to submit both the experimental group (post-gene-editing) and the control group (unedited) samples simultaneously, for accurate assessment of integration efficiency and elimination of background interference.

(1) Sample type and name;

(2) Gene-editing site information (gene name, chromosomal position);

(3) sgRNA sequence (including PAM sequence);

(4) Cut site coordinates (hg38 or other reference genome version);

(5) Donor sequence information (complete sequence with annotated homology arms, insertion sequence, and other key regions);

(6) Expected integration pattern description (single-copy/multi-copy, directional/non-directional, etc.);

(7) Editing type (single-site / dual-site / multi-site).

7.6 Value-Added Services

(1) Donor plasmid design and construction: design of optimal homology arms and insertion sequences according to integration strategy;

(2) sgRNA design and validation: provision of high-efficiency, low-off-target sgRNA sequences;

(3) Customized analysis: analysis content tailored to specific project characteristics;

(4) Regulatory submission technical support: provision of technical documents and data required for IND submission.

7.7 Important Notes

(1) All samples must meet the above quality standards to ensure the accuracy and reliability of detection results;

(2) For special sample types or non-standard requirements, please contact the ZhuHai GeneRulor technical team in advance;

(3) Contact: 400-6309596; Technical support email: service@generulor.com.

8. References

[1] Liu M, et al. (2021). Global detection of DNA repair outcomes induced by CRISPR–Cas9. Nucleic Acids Research, 49(15), 8732–8742.

[2] Liu Y, et al. (2022). PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair. STAR Protocols, 3(1), 101088.

[3] Yin J, et al. (2019). Optimizing genome editing strategy by primer-extension-mediated sequencing. Cell Discovery, 5, 18.

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

[5] FDA. Long Term Follow-Up After Administration of Human Gene Therapy Products – Guidance for Industry. 2020.

[6] National Medical Products Administration (NMPA) Pharmacopoeia Commission. Technical Guidelines for Pharmacological Research and Evaluation of In Vivo Gene Therapy Products (Trial). 2022.