Cat. No. / ID: 232103
CRISPR-Q Custom PCR Assays enable rapid and sensitive characterization of CRISPR-based genome editing events involving human, mouse or rat cells. These assays are designed for use with the QIAprep& CRISPR Kit with an innovative workflow that combines liquid-based sample preparation with end-point PCR detection 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.
The CRISPR-Q Custom PCR Assays and the QIAprep& CRISPR Kit provide sensitive detection from a wide cell input range (see figure The QIAprep& CRISPR Kit enables a broad range of cell inputs for PCR). Our advanced assay design algorithm provides full support for human, mouse and rat targets and generates robust primer sets that amplify the specified targets with a high success rate.
CRISPR-Q Custom PCR Assays allow you to characterize CRISPR editing events in cultured human, mouse or rat cells. The easy-to-use assay design tool is available in GeneGlobe: just enter your chromosome number, cut site and guide RNA sequence, and our advanced design algorithm will do the rest. It checks for the presence of gRNA sequence in the PCR product to ensure amplification of the correct region of interest. It also checks for off-target amplification to ensure that only the specific target is amplified and to provide an optimal user experience.
Three primer sets flanking the endonuclease cut site are designed for each target. The resulting PCR amplicons are 400–600 bp long with at least 150 bp from the cut site. The amplicons are compatible with various downstream analysis methods such as Sanger sequencing or mismatch detection assays.
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).
CRISPR-Q Custom PCR Assays can be directly used for target amplification form lysed cultured cells derived from human, mouse or rat without additional optimization of the PCR. The PCR primers are designed so that the resulting PCR product can be used for various downstream analyses.
Assays for amplifying genomic regions of interest
CRISPR-Q Custom PCR Assays can be easily designed and ordered for human, mouse or rat gene targets using the intuitive custom builder tool available in GeneGlobe at www.geneglobe.com/customize/crispr/ (see figure CRISPR assay design is optimized for human, mouse and rat gene targets). The custom builder tool generates several target-specific assays based on the genomic location and the sequence of the guide RNA (gRNA) 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:
(A) U2OS cell lysates were prepared according to the QIAprep& CRISPR Kit protocol. Cell input ranged from 0.5-25 cells/rxn (technical duplicates). (B) Jurkat cell lysates were prepared according to the QIAprep& CRISPR Kit protocol. Cell input ranged from 1,000-50,000 cells/rxn. The CRISPR-Q Custom PCR Assay targeting EMX1 was used for PCR. The presence of PCR product was verified using the QIAxcel Advanced.
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.