GeneCopoeia's IndelCheck™ CRISPR/TALEN insertion or deletion (indel) detection system provides a powerful means for assisting you in your genome editing applications. The IndelCheck™ system can be used for:
- Complete system to simplify your CRISPR/TALEN validation and edited clone screening
- Robust amplification for the target site PCR. No genomic DNA isolation is required
- Easy to use T7 endonuclease I assay with optimized conditions and positive control
Application 1: CRISPR sgRNA/TALEN Functional Validation
Figure 1. CRISPR or TALEN functional validation using the IndelCheck™ system. Cells transfected with CRISPR or TALEN plasmids are harvested in bulk, followed by generation of a PCR product using primers flanking the target site with the Target site PCR kits (1). The PCR product is denatured, followed by re-annealing, leading to a population of double strand fragments, some of which contain mismatches. These mismatches are detected by the T7 endonuclease I Assay kit (2). If CRISPR or TALEN are active in the cell, then cleavage products will be visible on an agarose gel.
Application 2: Screening for CRISPR- or TALEN-mediated genomic modifications
Figure 2. Using the IndelCheck™ system to screen for cell clones carrying desired CRISPR- or TALEN-mediated genomic modifications. Cells transfected with CRISPR or TALEN plasmids are plated for single clones, followed by genration of a PCR product using primers flanking the target site with the Target site PCR kits (1). In Protocol A (left) for identifying NHEJ-mediated knockouts, PCR products are screened for indels using the T7 Endonuclease I Assay kits (2). Positive PCR products are then cloned into a plasmid vector using the Target Site PCR Cloning kit (3) and sequenced to confirm the presence of the mutation(s). In Protocol B, for identifying any knockout or knockin modification, PCR products are cloned directly into a plasmid vector using the Target Site PCR Cloning kit (3) and sequenced to detect the presence of the mutation(s).
The figure below displays a typical validation result using the IndelCheck™ Target Site PCR kit and T7 Endonuclease I Assay kit:
|Figure 3. T7 Endonuclease I digestion of genomic amplicons modified by Cas9-sgRNA. Control cells (-) should only have a single band corresponding to uncut amplicon. Amplicons from sample cells (+) transfected with active Cas9-sgRNA yield 3 bands: 1 unmodified + 2 cleavage products of predicted sizes.|
The IndelCheck™ system consists of the following major components:
- Target site PCR kit V2.0. For amplification of targeted genomic regions from cell lysates without genomic DNA isolation. Now in Version 2.0, the Target Site PCR kit is an conains buffer, SuperHeRo™ DNA polymerase, and nucleotides together in one mix for greater convenience.
- T7 endonuclease I assay kit. For detection of CRISPR/TALEN-introduced indel mutations near target site(s).
- Target Site PCR cloning kit (coming soon). For cloning of PCR products to be used for sequencing and identification of CRISPR- or TALEN-mediated genomic modifications.
GeneCopoeia also offers CRISPR/TALEN validation services using the IndelCheck™ CRISPR/TALEN insertion and deletion detection system. We can validate the CRISPR/TALEN you purchased from us using HEK293 cells (human), NIH3T3 cells (mouse), or your specific cell line(s). Please contact us at email@example.com or 1-866-360-9531.
Material Safety Data Sheet
Fujimoto,D., et al. (2016). Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma. International Journal of Oncology doi: 10.3892/ijo.2016.3822 [IndelCheck™ T7 Endonuclease I]