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TEG-Seq CRISPR Off-Target Analysis
An unbiased whole genome method to detect off-target events
The transformative CRISPR-Cas9 technology has revolutionized the field of genome editing by allowing for the removal, addition or alteration of sections of the genome to create custom engineered cell lines. While this system can achieve high editing efficiencies, the CRISPR-Cas9 system can cleave the target DNA at unintended locations. Let our trusted, experienced developers of Invitrogen GeneArt CRISPR products help you monitor and detect off-target events in your custom-designed stable cell lines.
What is TEG-Seq?
TEG-Seq (tag-enriched GUIDE-Seq) is a next-generation sequencing method developed by Thermo Fisher Scientific scientist to address the sensitivity limitations of current next-gen sequencing methods. In this method 5’ phosphorylated primers are used for PCR amplification and differential marking of amplicons containing a double-stranded DNA tag (dsTag) that was inserted at double-stranded DNA breaks (DSB) sites. Significantly reducing nonspecific amplification and improved sensitivity of DSB detection.
Why examine for off-target events?
The CRISPR-Cas9 genome editing system is a complex of Cas9 nuclease and a target-specific guide RNA (gRNA) that induces a double-stranded DNA break at a desired location. However, lack of specificity leading to off-target cleavage events is still of concern. Off-target cleavage events at unintended locations could result in undesired phenotypes or loss of function gene activity, which is especially detrimental for therapeutic applications. Thus, accurate detection and monitoring of off-target events is an important step in any genome editing project.
Over the years, there have been several next-generation sequencing (NGS) based analysis methods to monitor and detect off-target cleavage events. Many of these methods rely on in vitro methods that perform genome editing outside of the cellular context. In cellulo detection methods have been developed, including GUIDE-Seq in which genome editing occurs within the native cellular environment. GUIDE-Seq (genome-wide, unbiased identification of double-stranded breaks (DSB) enabled by sequencing) is the most widely used method for the detection of off-target events. However, most of the currently available in cellulo detection methods are not sensitive enough to detect low-frequency off-target events due to higher non-specific target amplification.
Figure 1. Comparison of TEG-Seq and GUIDE-Seq. Read numbers for (A) HEK4 and (B) VEG1 loci were plotted from individual on-target events (red) and off-target events from TEG-Seq (blue) and GUIDE-seq (yellow). The total off-target events detected by TEG-Seq is 252 for HEK4 and 27 for VEG1, and the total off-target events detected by GUIDE-Seq is 132 for HEK4 and 21 for VEG1. The read number for an individual target is also higher in TEG-Seq than GUIDE-Seq with a similar level of HGS sequencing depth. Cas9 and gRNA were delivered using plasmid format.
TEG-Seq Service workflow and timeline
Our TEG-Seq service is offered as a simple add-on to any current CRISPR genome editing cell line engineering service. If you are generating CRISPR edited cell lines yourself, we provide you with dsTag for transfection and send us your cell pellet and we take it from there. In a matter of weeks, you will receive a comprehensive report detailing our findings. Our technical team is happy to address any questions along the way and your dedicated project manager will provide status updates so that you are update with the progress of your project.
Co-transfection of dsTag and customer’s gRNA of interest. We ship the dsTag to you or can be added to your custom genome editing project.
After editing reaction, cells are pelleted for shipment to our laboratory for genomic DNA extraction. Optional services are available to assess on-target editing efficiency prior to TEG-Seq analysis.
After genomic DNA extraction, DNA is sheared for downstream amplicon generation, enrichment and NGS library preparation.
Samples are sequenced to a depth of one million reads per sample.
Sequencing data is processed and analyzed for off-target events. Deliverable is a report listing identified off-targets. Optional amplicon sequencing to validated identified off-targets available.
Co-transfection of dsTag and customer’s gRNA of interest. We ship the dsTag to you or can be added to your custom genome editing project.
After editing reaction, cells are pelleted for shipment to our laboratory for genomic DNA extraction. Optional services are available to assess on-target editing efficiency prior to TEG-Seq analysis.
After genomic DNA extraction, DNA is sheared for downstream amplicon generation, enrichment and NGS library preparation.
Samples are sequenced to a depth of one million reads per sample.
Sequencing data is processed and analyzed for off-target events. Deliverable is a report listing identified off-targets. Optional amplicon sequencing to validated identified off-targets available.
Optional services
Choose from a number of additional services available to enable full customization of your off-target analysis or continue your project with downstream applications such as stable cell line generation and stem cell characterization analyses.
- NGS On-Target Analysis or Genomic Cleavage Detection Assay (GCD) to ensure editing occurred prior to TEG-Seq analysis
- Amplicon Sequencing to validate identified off-target events
Start your TEG-Seq project today
Contact us today to discuss your TEG-Seq project with a technical specialist.
Resources
For Research Use Only. Not for use in diagnostic procedures.