PCR-Based
On-/Off Target Analysis

Shearing Extension Primer Tag Selection Ligation-Mediated PCR (S-EPTS/LM PCR), a PCR-based approach for integration site analysis of gene therapy vectors, was adapted to analyze on-target and off-target integrations arising from genome editing. The standard S-EPTS/LM PCR protocol shears DNA resulting in ~500 bp fragments. For on-/off target analysis, the method was tailored to increase the shearing size to ~1000 bp.

Quantification of on-target integrations

To bridge the gap between the homology regions and to detect the sequences present after the homology regions, the first S-EPTS/LM-PCR extension primers are placed as close as possible to the homology regions in the target site and (5-10) predicted off-target sites, followed by asymmetric sequencing.

This allows us to evaluate:

  • Targeted integration of the donor construct
  • Integration frequency of ITR-AAV into the target site DSB
  • Knockout rate by indels at the target site
  • Indel and translocation characterization at the target site
  • Unmodified wildtype at the target site

Detection of random integrations

A similar SEPTS-PCR and asymmetric sequencing approach to bridge the homology regions and to detect the sequences present after the homology regions can be used to detect random integrations of the AAV vector (including integrations in target site), AAVS1, and any other overrepresented off-target locations of CRISPR/Cas9.

Check out our resources

CRISPR/Cas9:gRNA off-target analysis

Using locus-specific S-EPTS/LM-PCR, chosen predicted off-target sites can be analyzed and quantified.

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