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QIAprep&amp CRISPR Kits

For direct enhanced PCR amplification to confirm CRISPR editing events in human, mouse, or rat cells

Features

  • Fast and simple workflow allows rapid and sensitive characterization of CRISPR-based genome editing events
  • Compatible with adherent or suspension-cultured human, mouse or rat cells
  • Cell lysis preparation starting from down to 10 cells/µl 
  • Sensitive detection from a wide cell input range supports various DNA editing scenarios
  • Unique internal control detects and distinguishes sample or assay quality issues

Products

QIAprep&amp CRISPR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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QIAprep&amp CRISPR Kit (250)

Cat. No. / ID: 232101

For 250 x 20 μl reactions: 12 ml Cell Lysis Buffer, 100 μl Proteinase K, 1.25 ml AllTaq Master Mix (4x), 50 μl Master Mix Tracer (125x), 2 ml Q-Solution (5x), 100 μl CRISPR-Q Control PCR Assay, 2 x 1.9 ml RNase-Free Water
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QIAprep&amp CRISPR Kit (1000)

Cat. No. / ID: 232102

For 1000 x 20 μl reactions: 4 x 12 ml Cell Lysis Buffer, 4 x 100 μl Proteinase K, 4 x 1.25 ml AllTaq Master Mix (4x), 4 x 50 μl Master Mix Tracer (125x), 4 x 2 ml Q-Solution (5x), 4 x 100 μl CRISPR-Q Control PCR Assay, 8 x 1.9 ml RNase-Free Water

Product Details

The QIAprep&amp CRISPR Kit is an integral part or our QIAprep&amp CRISPR solutions workflow, which provides rapid and sensitive characterization of CRISPR-based genome editing events in adherent or suspension-cultured human, mouse or rat cells. The innovative system combines liquid-based sample preparation with downstream PCR detection of your region of interest using CRISPR-Q Custom PCR Assays and optional sequencing using CRISPR-Q Sanger Primers. The fast and simple workflow allows all-in-one processing and analysis in only a few steps and is compatible with different plate formats and cell types. The QIAprep&amp CRISPR Kit allows cell lysis preparation starting from down to 10 cells/µl and direct PCR detection from down to 1 cell. 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 QIAprep&amp CRISPR Kit provides sensitive detection from a wide cell input range (see figure  The QIAprep&amp CRISPR Kit enables a broad range of cell inputs for PCR).

The Cell Lysis Buffer provides efficient room-temperature lysis without affecting downstream target amplification (see figures  Cell Lysis Buffer rapidly lyses cells at room temperature and  Target amplification unaffected by cell lysis buffer). Sample lysis and amplification is also tolerant of residual amounts of common reagents such as cell growth media, culture dish coatings and transfection/transduction reagents (see table below and figure  Increased presence of medium might affect CRISPR target amplification). 

Substance tested Tolerated concentration
DMEM complete medium Up to 25% of lysate volume
Polybrene (Hexadimethrine Bromide) Up to 1 µg/ml*
Lipofectamine CRISPRMAX™ Concentration recommended by the manufacturer
Lipofectamine 2000® Concentration recommended by the manufacturer
DharmaFECT® Duo Concentration recommended by the manufacturer
Effectene® Concentration recommended by the manufacturer
Corning® Cell-Tak 3.5 µg/cm2 in culture vessel
Poly-L-Lysine 100 µg/ml
Fibronectin 10 µg/ml
Gelatin 0.1% (w/v)
Gelatin from cold water fish skin 2% (w/v)

* Final concentration in target amplification reaction that did not influence PCR outcome.
Concentration that was used during treatment of cells and that did not negatively influence cell processing and PCR amplification of targets.
Concentration used for coating of cell culture vessels prior cultivation of cells that did not affect cell lysate preparation and PCR amplification of targets.

See figures

Principle

The QIAprep&amp CRISPR Kit along with CRISPR-Q Custom PCR Assays and CRISPR-Q Sanger Primers allows you to characterize CRISPR editing events in cultured human, mouse or rat cells in only four steps: liquid-based sample preparation, custom primer design, amplification of the region of interest and quantification of editing efficiency. The CRISPR-Q Sanger analysis tool, which is available in GeneGlobe, enables calculation and visualization of 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).

Cell processing and sample preparation

Cultured CRISPR-edited cells are briefly washed to remove cell culture medium, extracellular material released by living cells and intracellular material released by any dead, lysed cells. Removal of this material is recommended, since it can interfere with downstream sample preparation and amplification processes. For the preparation of CRISPR-edited genomic DNA, the Cell Lysis Buffer supplemented with Proteinase K is directly added to the cells.

Cell lysis

The Cell Lysis Buffer included in the QIAprep&amp CRISPR Kit is optimized for efficient cell lysis, increased lysate stability and high compatibility with the AllTaq PCR chemistry. The lysis reaction can accommodate a broad range of cell numbers and takes place either in a tube or in the culture plate at room temperature. The lysis is stopped at 80°C, and the raw cell lysate can be directly used as input DNA for the PCR reaction with no intermediary purification steps.

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.

PCR amplification of genomic regions of interest

PCR amplification of the genomic region of interest can be used to assess the success of the CRISPR gene editing event in the cultured cells. In addition to efficient cell lysis, the QIAprep&amp CRISPR Kit also provides a highly compatible PCR chemistry: the AllTaq PCR chemistry allows reliable amplification of target genes from raw cell lysates produced with the Cell Lysis Buffer and CRISPR-Q Custom PCR Assays or other PCR primers.

AllTaq Master Mix

The AllTaq Master Mix provides a convenient format for highly sensitive and specific hot-start PCR using any DNA template. The ready-to-use Master Mix contains the AllTaq DNA Polymerase, AllTaq PCR Buffer and dNTPS and is provided as a 4x concentrate, which allows for a higher sample input.

Q-Solution

Q-Solution is an additive that facilitates amplification of difficult templates by modifying the melting behavior of nucleic acids. Q-Solution often enables or improves suboptimal PCR caused by DNA templates that have a high degree of secondary structure or that are GC-rich.

Master Mix Tracer

The Master Mix Tracer is an orange dye that enables visual tracking during PCR setup and serves as a loading dye for agarose gels. The dye runs at approximately 50 bp on a 1% agarose gel. The 125x concentrate can either be added to the PCR reaction mix or directly to the master mix stock vial to obtain a 1x final concentration.

CRISPR-Q Control PCR Assay

The CRISPR-Q Control PCR Assay is provided as a 20x concentrate and can be used as a positive control in the PCR (see figure  The QIAprep&amp CRISPR Kit includes a PCR control to help determine lysate quality). It amplifies a conserved target region in human, mouse and rat and alerts you to issues such as insufficient DNA input or insufficient lysate quality. The size of the control PCR product is 261 bp.

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 available in GeneGlobe at www.geneglobe.com/customize/crispr/. This tool generates several target-specific primers based on the genomic location and the sequence of the (gRNA) used for your particular CRISPR gene editing events (see figure  The CRISPR-Q Sanger Primers successfully sequence target regions of interest).

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

Quick-Start Protocols (1)
Brochures & Guides (1)

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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