CRISPR-Q Sanger Primers

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

Features

  • Highly specific primers for successful sequencing and analysis
  • Simple-to-use primer design tool requires no design expertise
  • Sanger analysis tool provides fast and easy analysis of sequencing traces and calculation of editing efficiency
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CRISPR-Q Sanger Primers (10 µM)

Cat. No. / ID: 232104

Two vials containing lyophilized Sanger sequencing primers for analyzing PCR products generated with the corresponding CRISPR-Q Custom PCR Assay; 10 µM
A$85.20
 CONFIGURE AT GeneGlobe
CRISPR-Q Sanger Primers are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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&amp 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.

Performance

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). 

 

See figures

Principle

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.

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). First, the target region is amplified using the QIAprep&amp 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.

 

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

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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