PE-Tag Off-Target Detection for Prime Editing

PE-Tag Off-Target Detection for Prime Editing

1. Background

With the widespread clinical application of CRISPR and other gene-editing technologies, safety evaluation has become increasingly important. The U.S. Food and Drug Administration (FDA) has explicitly required comprehensive assessment of potential off-target effects induced by gene-editing products. As a precision gene-editing technology that does not rely on DNA double-strand breaks (DSBs), Prime Editor (PE) offers the advantages of high precision and high specificity; however, its potential off-target effects still require systematic evaluation.

The Primer-Extension-Mediated Tagging Sequencing (PE-Tag) gene-editing off-target detection platform developed by ZhuHai GeneRulor enables genome-wide precise capture and analysis of Prime Editor editing activity based on NGS technology. This technology introduces a specific tag sequence at editing sites, combined with high-throughput sequencing, to comprehensively detect editing activity of the PE system across the entire genome, providing key technical support for scientific research and preclinical safety evaluation. Based on their different research needs, clients can flexibly select from two detection strategies: in vitro PE-Tag — using purified DNA for high-sensitivity comprehensive screening — and in cells PE-Tag — simulating a true biological environment to ensure the biological relevance of off-target analysis results.

2. PE-Tag Detection Principle and Flexible Detection Strategies

PE-Tag is a highly sensitive, genome-wide detection method for Prime Editor editing activity. Its fundamental principle involves introducing specific tag sequences at editing sites via Prime Editor, followed by tag-specific amplification and high-throughput sequencing to achieve genome-wide detection of PE editing sites. ZhuHai GeneRulor offers two detection strategies — In vitro PE-Tag and In cells PE-Tag — to meet different research needs:

2.1 In vitro PE-Tag (Purified DNA-Level Detection)

(1) Principle: purified PE2 protein and tag-sequence-containing pegRNA are used to process extracted genomic DNA in vitro, followed by tag-specific amplification and high-throughput sequencing to detect editing sites;

(2) Advantages: high sensitivity, low background, capable of detecting more low-frequency off-target sites; not limited by cell transfection efficiency;

(3) Applicable scenarios: projects with high sensitivity requirements for off-target detection, preliminary screening of potential off-target sites, or samples that cannot be readily used for cell transfection.

Figure 1. Principle of the In vitro PE-Tag library construction workflow

2.2 In cells PE-Tag (Cell-Level Detection)

(1) Principle: PE2 protein and pegRNA are introduced into live cells via cell transfection or electroporation to allow editing to occur intracellularly, more closely approximating real biological conditions;

(2) Advantages: reflects the true editing situation in a cellular environment, accounting for the influence of cellular barriers, chromatin status, and other factors;

(3) Applicable scenarios: validation of high-confidence off-target sites, simulation of clinical application scenarios, and assessment of editing specificity in complex biological environments.

Figure 2. Principle of the In cells PE-Tag library construction workflow

2.3 PE-Tag Library Construction Workflow

The PE-Tag library construction workflow primarily includes:

(1) Introduction of editing system: deliver the PE system (protein, mRNA, or plasmid form) together with tag-sequence- and target-sequence-containing pegRNA into the reaction system (in vitro) or into cells (in cells);

(2) Tag integration: introduce specific tag sequences at active sites (including on-target and off-target sites);

(3) Fragmentation: use Tn5 transposase to fragment the tagged genomic DNA, simultaneously ligating adapter sequences containing unique molecular identifiers (UMI);

(4) PCR specifically amplify DNA fragments containing tag sequences;

(5) Perform high-throughput sequencing on the amplified products;

(6) Apply a professional bioinformatics analysis pipeline to precisely identify all PE editing sites across the genome.

3. PE-Tag Detection and Analytical Advantages

3.1 Comprehensive Capture of Prime Editor Editing Activity

PE-Tag technology enables efficient detection of the following categories of Prime Editor editing events:

(1) On-target editing: precise detection of editing efficiency and editing patterns at target sites;

(2) Off-target editing: comprehensive genome-wide capture of potential off-target sites;

(3) pegRNA design evaluation: assessment of the specificity and off-target risk associated with different pegRNA designs.

3.2 Technical Features and Advantages

(1) High specificity: specific capture of Prime Editor active sites, reducing false-positive rates;

(2) Genome-wide coverage: independent of bioinformatic predictions; truthfully reflects editing system activity across the entire genome;

(3) Flexible detection strategies: detection mode (in vitro or in cells) can be selected based on project needs, balancing sensitivity and biological relevance;

(4) Broad sample type compatibility: applicable to purified DNA, cell samples, and in vivo samples;

(5) Direct detection: detection based on actual Prime Editor activity, rather than indirect inference;

(6) Multi-PE system compatibility: applicable to evaluation of multiple Prime Editor systems including PE2, PEmax, and epegRNA.

3.3 Service Advantages

(1) Customized detection plans: the most suitable PE-Tag detection strategy (in vitro or in cells) is provided based on research objectives, sample type, and editing system;

(2) Full-panel evaluation: comprehensive PE editing profile analysis covering both on-target and off-target sites;

(3) Multi-layer analysis: comprehensive molecular-level assessment of gene-editing off-target effects;

(4) Flexible applicability: suitable for both basic scientific research and preclinical safety evaluation;

(5) Professional report: provision of a comprehensive analytical report covering editing efficiency, off-target site analysis, and functional annotation;

(6) Data interpretation: professional data interpretation and technical consultation to assist experimental design and result analysis.

4. Technical Application Scenarios

4.1 Application Fields

（1）Basic research applications: supporting optimization and improvement of Prime Editor systems in academic research;

（2）pegRNA design optimization: screening for pegRNA sequences with the highest specificity and lowest off-target risk;

（3）Preclinical safety evaluation: providing data support for safety assessment of gene-editing therapeutics;

（4）Editing system comparison: evaluating the editing specificity of different Prime Editor systems (e.g., PE2, PEmax, epegRNA, etc.);

（5）Target site safety assessment: evaluating potential off-target risks across different therapeutic target sites;

（6）Drug development support: providing key safety data for gene therapy drug development.

5. PE-Tag Detection Report Content

The PE-Tag analytical reports provided by ZhuHai GeneRulor adopt a standardized format to ensure professional and comprehensive data presentation. The introductory section covers project background, technical principles, PE-Tag library construction workflow description, and bioinformatics analysis pipeline overview, providing clients with the necessary technical context. This is followed by sample sequencing data statistics, which include detailed sample data quality information and alignment results to ensure data quality and reliability. In addition, the report includes the following key components:

(1) Off-Target Site Identification Results: the system displays potential off-target sites identified by PE-Tag, including key data such as off-target site positional information, site sequence, alignment with the pegRNA sequence, and UMI count.

Figure 3. PE-Tag representative report (off-target site identification)

(2) Off-Target Sequence Alignment Visualization: through an intuitive sequence alignment map, the similarity and differences between off-target sites and the pegRNA sequence are displayed, with key mismatch positions annotated, helping clients intuitively understand the sequence characteristics and underlying causes of off-target sites.

Figure 4. PE-Tag representative report (off-target sequence alignment visualization)

(3) Off-Target Site Functional Annotation: functional annotation analysis is performed for each identified off-target site, including the nearest gene, positional characteristics (exonic, promoter region, etc.), and whether cancer-associated genes are involved and other key information, enabling assessment of the potential biological impact of off-target events.

Figure 5. PE-Tag representative report (off-target site functional annotation)

6. PE-Tag Service Content

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

* Service highlight: integrated end-to-end service support from pegRNA design through final data analysis is available; in vitro or in cells detection strategy can be flexibly selected based on research needs.

7. PE-Tag 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: >2×10⁵ 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] Liang SQ, Liu P, et al. (2023). Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag. Nature Methods, 20(5), 738–748.

[2] Anzalone AV, et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157.

[3] Chen PJ, et al. (2021). Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635–5652.

[4] Nelson JW, et al. (2021). Engineered pegRNAs improve prime editing efficiency. Nature Biotechnology, 40(3), 402–410.

[5] Liu P, et al. (2021). Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nature Communications, 12(1), 2121.

[6] Gao R, et al. (2022). Genomic and transcriptomic analyses of prime editing guide RNA-independent off-target effects by prime editors. CRISPR Journal, 5(2), 276–293.