Cat. No. / ID: 232104
CRISPR-Q Sanger Primers enable rapid characterization of CRISPR-based genome editing events involving human, mouse or rat cells. These primers are designed for use with the QIAprep& CRISPR Kit with an innovative workflow that combines liquid-based sample preparation with Sanger sequencing-based analysis of your region of interest. The CRISPR-Q Sanger Sequencing Analysis tool, which is available in GeneGlobe, enables quick and convenient analysis to complete the entire CRISPR editing detection workflow.
CRISPR-Q Sanger Primers and the corresponding CRISPR-Q Sanger Sequencing Analysis Tool allow you to calculate and visualize the efficiency of your editing event with speed, convenience and accuracy (see figure The CRISPR-Q Sanger Primers successfully sequence target regions of interest).
The easy-to-use CRISPR assay design tool, which is available in GeneGlobe, requires no primer design expertise and provides full support for human, mouse and rat targets. Just enter your chromosome number, cut site and guide RNA sequence, and our advanced design algorithm will do the rest. The design is based on the genomic location of the cut and generates three robust PCR primer sets and corresponding CRISPR-Q Sanger Primers that enable you to analyze the specified region with high success. The CRISPR-Q Sanger analysis tool, also available in GeneGlobe, enables calculation and visualization of your CRISPR editing events.
The QIAprep& CRISPR Kit offers a streamlined workflow, which combines a liquid-based sample preparation step that can be completed in only 25 minutes with sensitive PCR and Sanger sequencing detection (see figure The QIAprep& CRISPR Kit workflow). First, the target region is amplified using the QIAprep& CRISPR Kit and CRISPR-Q Custom PCR Assays. Following purification of the PCR product, it is sequenced using the CRISPR-Q Sanger Primers and the sequence traces are analyzed using the CRISPR-Q Sanger Sequencing Analysis tool, which is available in GeneGlobe.
Primers for Sanger sequencing of genomic regions of interest
CRISPR-Q Sanger Primers can be easily designed and ordered using the intuitive custom builder tool, which is also available in GeneGlobe. This tool generates several target-specific primers based on the genomic location and the sequence of the guide RNA used for your particular CRISPR gene editing events.
The QIAprep& CRISPR Kit and corresponding CRISPR-Q Custom PCR Assays and CRISPR-Q Sanger Primers are well-suited for practically any researcher who needs to characterize CRISPR-based editing events:
The new CRISPR workflow combines fast, liquid-based sample preparation with PCR and Sanger sequencing-based analysis for checking the gene editing efficiency.
Ten cells per microliter cell lysis buffer are sufficient. This significantly cuts cultivation time and speeds up the gene edit characterization.
Yes. All editing events that are covered for CRISPR are also covered for TALENs and ZFN.
We recommend sequencing from both directions (5' and 3'). However, the forward and the reverse traces are analyzed separately. What is needed is a control trace (e.g., from WT) that is compared to a sample trace (edited sample). The control sample is crucial. Without a control trace, the calculation of the editing efficiency is not working. Additionally, the gRNA sequence used for editing without PAM is needed.
CRISPR-Q Custom PCR Assays are designed in a way that the resulting PCR product can be analyzed by T7 endonuclease assays or similar methods and Sanger sequencing.
The AllTaq PCR chemistry included in the QIAprep& CRISPR Kit is very robust against inhibitors. The sample preparation is not affected, and the PCR reaction tolerates up to 1 µg/ml.
The CRISPR-Q Custom PCR Assays and the CRISPR-Q Sanger Primers are in silico validated with the design algorithm on GeneGlobe. Assays and primers are not wet-bench validated.
Raw lysate is okay with PCR; there's no need to purify the lysate. Only the PCR product needs to undergo purification before Sanger sequencing.
The CRISPR-Q Custom PCR Assay is not for dPCR use. It is for a conventional PCR run in a standard cycler, and the PCR product is analyzed on the QIAxcel or a gel. The CRISPR-Q Custom PCR Assay contains 2 primers flanking the cut site. The purpose is to do a quick PCR check of the clones as well as preparation of template for the following Sanger sequencing. For Sanger sequencing, the corresponding CRISPR-Q Sanger Primers are used. dPCR for checking the editing event is another option to Sanger Sequencing and would be specific for the event. Such a dPCR Assay Product is in progress and launch is planned for the end of 2021.
The QIAprep& CRISPR sample preparation and target amplification are not affected by coating reagents. Additional information and a list of tested coating reagents can be found in the QIAprep& CRISPR and CRISPR-Q Custom Kits Handbook in Appendix C.