PEM-seq Chromosomal Rearrangement Detection

PEM-seq Chromosomal Rearrangement Detection

1. Background

With the widespread clinical application of CRISPR and other gene-editing technologies, their potential safety risks have drawn increasing attention from regulatory agencies worldwide. DNA double-strand breaks (DSBs) generated by CRISPR cleavage can lead to chromosomal rearrangement events during the repair process, including large chromosomal deletions, insertion of exogenous DNA fragments, and chromosomal translocations. The U.S. Food and Drug Administration (FDA) has explicitly emphasized the importance of chromosomal rearrangement detection, as these events may cause serious adverse outcomes including genomic instability, elevated disease risk, treatment failure, and other unpredictable consequences [1–4].

Figure 1. FDA guidance document Human Gene Therapy Products Incorporating Human Genome Editing: key recommendations on the detection of chromosomal structural abnormalities

The Primer-Extension-Mediated Sequencing (PEM-seq) chromosomal rearrangement detection platform, launched by ZhuHai GeneRulor in 2022, enables highly sensitive quantification of gene-editing-induced genomic structural variants based on next-generation sequencing (NGS) technology. To ensure technical reliability and accuracy, the ZhuHai GeneRulor team has conducted comprehensive method validation in strict accordance with ICH Q2(R1) guidelines and the FDA Guidance for Industry on Analytical Procedures and Methods Validation for Drugs and Biologics, establishing a complete technical evaluation framework. ZhuHai GeneRulor has successfully provided gene-editing safety evaluation services compliant with regulatory standards to numerous domestic and international gene therapy companies, fully supporting IND submissions and clinical translation.

2. Detection Principle of PEM-seq

PEM-seq is a highly sensitive chromosomal rearrangement detection method capable of precisely identifying editing-induced chromosomal rearrangement events [4]. The underlying principle involves the use of biotinylated bait primers (lure primers) designed upstream or downstream of the DSB site to capture sequences that rejoin at the DSB locus following repair (prey sequences). The detailed detection workflow comprises the following steps:

1. Sonicate genomic DNA to fragments of 300–700 bp;

2. Perform linear extension using biotinylated primers located 50–110 bp from the cut site;

3. Capture single-stranded, biotin-labeled DNA using streptavidin-coated magnetic beads;

4. Ligate a bridge adapter containing random molecular barcodes at the 3’ end to enable exponential amplification of target fragments;

5. Perform nested PCR with I5-Nested and I7 index primers followed by library amplification using Illumina adapter primers with unique index tags;

6. Sequence the library by high-throughput sequencing and analyze sequence variations in the target region.

Figure 2. Schematic illustration of the PEM-seq detection principle

3. Detection and Analytical Advantages of PEM-seq

3.1 Comprehensive and Precise Detection of Multiple Gene-Editing Rearrangement Events

PEM-seq technology enables efficient detection of the following categories of gene-editing-induced chromosomal rearrangement events:

1. Single-site editing rearrangements: precise detection of large deletions, insertions, and translocations with other genomic loci in the vicinity of a single cut site;

2. Multi-site editing rearrangements: quantification of chromosomal rearrangement risk between different on-target cut sites during simultaneous multi-site editing;

3. Off-target site rearrangements: identification of rearrangement events between off-target cut sites or between off-target and on-target/other genomic loci;

4. Background DSB translocation detection: detection of translocation events between background DSBs — arising from DNA replication or other physicochemical processes — and editing sites.

3.2 Outstanding Analytical Performance

ZhuHai GeneRulor rigorously follows ICH Q2(R1) [1] and FDA Bioanalytical Method Validation guidelines [2], conducting systematic validation of PEM-seq technology across four critical performance dimensions:

3.3 Service Advantages

●   Comprehensive method validation: PEM-seq has undergone systematic validation covering the full 0.001%–50% linear dynamic range with demonstrated high sensitivity and specificity;

●   Regulatory compliance assurance: strictly adheres to ICH Q2(R1) and FDA guidance requirements, ensuring detection results meet international regulatory standards;

●   Leading technology platform: PEM-seq enables comprehensive detection of multiple editing-induced chromosomal structural variant events, including Translocation, large-scale Deletion, Insertion, and Inversion, fully supporting safety evaluation requirements across all stages of drug development;

●   Rich success case portfolio: services have been successfully delivered to numerous gene therapy companies including Iaso Bio, Qinheng Bio, and BoYuan Bio, supporting smooth IND submissions;

●   Customized analytical solutions: customized detection schemes based on target site characteristics, with comprehensive analytical reports covering accuracy, sensitivity, reproducibility, and linearity;

●   Full-process service model: end-to-end gene-editing safety solutions encompassing target site safety pre-assessment, sample testing, and IND submission data support.

4. Application Scenarios of PEM-seq Detection

4.1 Application Fields of PEM-seq Technology

1. Target site screening-stage safety evaluation: molecular-level quantification of rearrangement risk to assist research teams in identifying optimal editing targets;

2. sgRNA design optimization: assessment of chromosomal rearrangement risk associated with different sgRNAs to guide selection of the safest sequence design;

3. Preclinical safety evaluation: fulfilling regulatory agency requirements for rigorous assessment of chromosomal rearrangement risk for gene-editing therapeutics;

4. IND submission data support: providing method validation reports and safety evaluation data compliant with ICH and FDA requirements;

5. Clinical sample monitoring: gene-editing safety monitoring for patient samples during clinical trials;

6. Production quality control: serving as a key quality indicator for the release of gene therapy products.

4.2 Representative Success Cases

ZhuHai GeneRulor has continuously provided comprehensive gene-editing safety evaluation services — encompassing PEM-seq detection, data analysis, and regulatory document preparation — to companies including Iaso Bio, Qinheng Bio, and BoYuan Bio, supporting the successful FDA IND submissions and regulatory approval of their CAR-T products. In addition, we have provided gene-editing safety evaluation services to numerous domestic and international gene therapy companies, supporting IND submissions and clinical translation of multiple products.

5. Representative PEM-seq Detection Report

The PEM-seq analytical reports provided by ZhuHai GeneRulor adopt a standardized format to ensure professional and comprehensive data presentation. The introductory section covers project background, experimental rationale, PEM-seq 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 (sample ID, experimental/control group classification, sgRNA sequences, and genomic coordinates), raw sequencing data quality metrics, and comparative reference genome alignment results, ensuring data quality and reliability. In addition, the report includes the following key components:

(1) DSB site editing event statistics: comprehensive statistics of all DSB site editing events, including Germline (unedited), Substitution, Deletion, Insertion, Inversion, and Translocation events, with precise counts and proportional analysis for each category. Visual representations are provided to display the genomic distribution of each event type across chromosomes.

Figure 3. PEM-seq representative report: statistics of editing events captured at the DSB site (left) and read-level visualization at the target site (right)

(2) Precise quantification of chromosomal translocation and inversion events: translocation and inversion events are classified by type (On-target + On-target, On-target + Off-target, and On-target + Background), with detailed records of chromosomal location, cut site coordinates, gene regions and functional impact of each event. Gene ontology annotation is also provided, with pie charts and tables showing the distribution and frequency of different functional impact categories, supplying scientific evidence for assessing the potential cellular impact of gene editing.

Figure 4. PEM-seq representative report: statistics and classification of translocation and inversion events

(3) Off-target site identification and evaluation: a dedicated algorithm is used to systematically identify and evaluate potential off-target sites, providing chromosomal coordinates, sequence alignment, functional annotation, and genomic distribution visualization (Circos plot) for each off-target site — delivering comprehensive analysis of editing specificity and potential off-target risks, with critical technical support for the safety evaluation of gene therapy products.

Figure 5. PEM-seq representative report: off-target site identification and characterization

6. PEM-seq Service Content

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

* Service highlight: integrated end-to-end service support from sgRNA design through final data analysis is available.

7. PEM-seq 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 cells for DNA extraction; cell quantity requirements: ≥2×10⁷ 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] Hu J, et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11, 853–871. doi:10.1038/nprot.2016.043 (2016).

[4] Yin J, et al. Optimizing genome editing strategy by primer-extension-mediated sequencing. Cell Discov 5, 18. doi:10.1038/s41421-019-0088-8 (2019).

[5] Yin L, et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing. Nat Commun 13, 1204. doi:10.1038/s41467-022-28900-w (2022).