Cat. No. / ID: 232101
Cat. No. / ID: 232102
The QIAprep& CRISPR Kit is an integral part or our QIAprep& 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& 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.
The QIAprep& CRISPR Kit provides sensitive detection from a wide cell input range (see figure The QIAprep& 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‡|
|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.
The QIAprep& 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.
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).
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.
The Cell Lysis Buffer included in the QIAprep& 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 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& 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 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& 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).
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:
The new CRISPR workflow combines fast, liquid-based sample preparation with PCR and Sanger sequencing-based analysis for checking the gene editing efficiency.
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.