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

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 .

The Cell Lysis Buffer provides efficient room-temperature lysis without affecting downstream target amplification . 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 . 

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.

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/ . 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. 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 .

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)