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CRISPR/Cas9 technology resources

CRISPR/Cas9 Technology Resources

GenEZ™ Next-Generation Molecular Cloning

Resources for performing CRISPR/Cas9 genome editing in your own laboratory:

CRISPR/Cas9 FAQs

1. What is CRISPR?

2. How are gRNA sequences designed?

3. How efficient and specific is CRISPR-mediated genome editing with gRNA constructs designed by GenScript?

4. What expression vector should I use?

5. What promoter should I use to drive gRNA expression?

CRISPR/Cas9 Experimental Protocol for genome editing in mammalian cell lines

The following procedure is based on HEK293 cells. To use host cell lines other than HEK293, please follow the instruction from original supplier for cell culture, passing the cells, transfection and subcloning. If you have specific questions about how to adapt this protocol for your needs, we recommend consulting the public online forum for Genome Engineering using CRISPR/Cas Systems.

Experimental Protocol for CRISPR/Cas9 genome editing to knock out a target gene

Experimental outline for knocking out a coding sequence in a mammalian cell line:

  • I. Host cell preparation

    Culture the host cells (HEK293) in Eagle's Minimum Essential Medium supplied with fetal bovine serum to a final concentration of 10%. Incubate cultures at 37°C.
    Subculture when cell concentration is between 6 and 7 x 104 cells/cm2.
    Seed 4-6x104 cells/cm2 in cell culture plate one day before transfection.

  • II. Transfection

    Transfect gRNA and cas9 into HEK293 cells using standard methods for HEK293 transfection. (Multiple transfection reagents give high transfection efficiency for HEK293 cells, following instructions from suppliers.)

    DNA ratio for the two elements for CRISPR-cas9 system

    Two vector systemAll-in-one vector
    gRNA++
    Cas9++
    Starting Ratio of plasmids1:1NA

  • III. Analysis of transfected cell pool

    2-3 days after transfection, the cell pool can be analyzed directly by Sanger sequencing, NGS (Next Generation Sequencing) and/or Surveyor assay. Here the commonly used methods of Sanger sequencing and Surveyor assay (or T7E1 assay) are briefly introduced.

    1. Sanger sequencing of the target region can detect overlapping peaks at close region of double strand break (DSB), if small insertion or deletion (indel) mutations are introduced.
    2. Surveyor assay (or T7E1 assay) uses enzymes of mismatch-specific DNA endonucleases to detect indel mutations at the targeted loci. By targeting and digesting mismatched heteroduplex double-strand DNA, this assay produces two or more smaller fragments, depending on number of mismatched sites on the region analyzed. These assays can be conducted following instructions from supplier.

  • IV. Cloning to obtain isogenic cell lines

    Transfected cells can be selected using antibiotic resistance or a GFP reporter if they are present on the Cas9 expression plasmid.*
    Transfected cells (with or without selection) can be plated into 96 well plate at 1 cell/well density for cloning. This procedure can be also conducted using diluted host cell line on 10 cm plate to form colonies, which can be picked up and transferred to 24 well plate for future usage.

    *Note, selection using antibiotic containing medium can induce random integration of the cas9 expression plasmid onto host genome.

  • sequencing to identify CRISPR-edited cell lines harboring desired mutationV. Screening for cell lines with desired mutation

    After expansion of the clones, the cells in each clone can be analyzed by Sanger sequencing to identify the clones harboring a mutation at the target region. Sequencing trace files will show overlapping peaks at regions where double strand breaks have been repaired by introducing small indels.

  • VI. Knock out cell line confirmation

    Knock-out cell lines can be confirmed by Western Blot if a specific antibody is available, or through functional assays specific to the gene that was targeted.

CRISPR/Cas9 Related Services

Legal Statement of GenCRISPR Services and Products (Updated on July 28, 2015):

  1. GenCRISPR™ services and products are covered under US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616 and foreign equivalents and licensed from Broad Institute, Inc. Cambridge, Massachusetts.
  2. The products and the reagents generated from these services shall be used as tools for research purposes, and shall exclude (a) any human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) any human germline modification, including modifying the DNA of human embryos or human reproductive cells, (c) any in vivo veterinary or livestock use, or (d) the manufacture, distribution, importation, exportation, transportation, sale, offer for sale, marketing, promotion or other exploitation or use of, or as, a testing service, therapeutic or diagnostic for humans or animals.
  3. The purchase of the GenCRISPR Services and Products coveys to the purchaser the limited, non-transferable right to use the products purchased and the reagents generated from GenCRISPR services and any related material solely for Research Purposes only, not for any Commercial Purposes.

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