CRISPR-Q Custom PCR Assays

For PCR-based characterization and confirmation of CRISPR editing events in human, mouse, or rat cells

Features

  • Optimized for usage with the QIAprep&amp CRISPR Kit
  • Fully customized for your region of interest
  • in silico validation ensures high success rate
  • Design available for human, mouse and rat genomes
  • PCR product obtained is compatible with T7 and Sanger sequencing analysis
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CRISPR-Q Custom PCR Assay (250)

Cat. No. / ID: 232103

One vial containing lyophilized custom PCR primer mix for 250 reactions of 20 µl each
$70.00
 CONFIGURE AT GeneGlobe
CRISPR-Q Custom PCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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&amp 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.

Performance

The CRISPR-Q Custom PCR Assays and the QIAprep&amp CRISPR Kit provide sensitive detection from a wide cell input range (see figure  The QIAprep&amp 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.

See figures

Principle

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.

Procedure

The QIAprep&amp 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&amp 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.

See figures

Applications

The QIAprep&amp 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:

  • Characterization of gene editing events
  • Functional studies including gene knock-out or insertion
  • Base editing
  • Genome screening (CRISPRi libraries, reversible knockdowns)

Supporting data and figures

Resources

FAQ

What is the minimum cell number needed for cell lysis?

Ten cells per microliter cell lysis buffer are sufficient. This significantly cuts cultivation time and speeds up the gene edit characterization.

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Is the QIAprep& CRISPR Kit also applicable to other gene-editing technologies such as TALENs and ZFN?

Yes. All editing events that are covered for CRISPR are also covered for TALENs and ZFN.

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Which database versions are used for CRISPR-Q Custom PCR Assay and CRISPR-Q Sanger Primers design?

Human: GRCh38
Mouse: GRCm39
Rat: mRatBN7

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For the Sanger analysis tool, the customer needs to upload two .abi1 files of forward and reverse sequences? Is there any additional info that needs to be provided?

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.

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Are the PCR products generated with CRISPR-Q Custom PCR Assays only applicable to analysis of editing efficiency by Sanger sequencing?

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.

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Is there something to consider when working with transduced cells treated with Polybrene?

The AllTaq PCR chemistry included in the QIAprep&amp CRISPR Kit is very robust against inhibitors. The sample preparation is not affected, and the PCR reaction tolerates up to 1 µg/ml.

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Are the CRISPR-Q PCR Assays and the CRISPR-Q Sanger Primers on GeneGlobe validated?

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.

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For sequencing application, does the lysate require purification?

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.

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Can the new CRISPR PCR Assays be used for dPCR on the QIAcuity? Do you have data or a protocol?

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.

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Is there anything to consider when processing cultured cells on coated cultivation dishes? Are additional purification steps required to eliminate or reduce coating reagents such as Poly-L-Lysine?

The QIAprep&amp 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&amp CRISPR and CRISPR-Q Custom Kits Handbook in Appendix C.

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