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CRISPR-Cas9 knockout protocols are designed to generate precise gene knockouts by introducing targeted mutations through double-stranded DNA breaks, followed by error-prone repair mechanisms like non-homologous end joining (NHEJ). Below is a typical step-by-step CRISPR-Cas9 knockout protocol, covering the design, delivery, and validation stages.

 CRISPR-Cas9 Knockout Protocol

 Materials

  1. Cell line: Choose the cell line you want to modify (e.g., HEK293T, CHO, etc.).
  2. Plasmids:

Cas9 expression vector (or a cell line that stably expresses Cas9).

gRNA expression vector (e.g., in plasmid form).

Optional: Repair template if using homology-directed repair (HDR) for precise modifications.

  1. Lipofection reagent: For transfection (e.g., Lipofectamine, PEI).
  2. Selection antibiotic: If using selection markers (e.g., puromycin, G418).
  3. Primers: For PCR validation of gene editing.
  4. Next-generation sequencing (NGS) services or sequencing tools: For validation of mutations.
  5. qPCR reagents: For expression analysis if needed.
  6. Antibodies: For protein expression validation, if necessary.

Procedure

Design and Selection of gRNA

Design gRNAs: Design guide RNAs (gRNAs) that specifically target the gene of interest. Most protocols use online tools like:

CRISPR design tools from the Broad Institute (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)

Benchling or CHOPCHOP

Target Exon(s): Choose gRNAs targeting an essential exon in the gene to ensure a knockout. The gRNA should direct Cas9 to create a double-strand break (DSB).

Target sequences should have a 5’-NGG-3’ PAM (protospacer adjacent motif) sequence.

Off-Target Analysis: Ensure minimal off-target effects by selecting gRNAs with high specificity using design tools.

Cloning of gRNA into Expression Vector

Clone the selected gRNA sequence into a plasmid vector expressing both the gRNA and Cas9, or into separate plasmids.

Vectors like pSpCas9(BB)-2A-Puro (PX459) from Addgene are commonly used.

Optional: Use a plasmid with a fluorescent marker (e.g., GFP) for transfection efficiency tracking.

Transfection of Cells

Seed Cells: Seed the cells in a 6-well plate or another appropriate format so that they are 50-70% confluent at the time of transfection.

Transfection: Transfect the cells with the gRNA-Cas9 plasmid (and optionally, a donor template for HDR) using a lipofection reagent (e.g., Lipofectamine or PEI) or electroporation.

Amount of DNA: Use 1-2 µg of plasmid DNA per well of a 6-well plate.

Incubation: Incubate the cells for 24-48 hours to allow for gRNA expression and Cas9-mediated DNA cleavage.

Selection of Transfected Cells (Optional)

Antibiotic Selection: If your plasmid contains an antibiotic resistance gene (e.g., puromycin or G418 resistance), apply the appropriate antibiotic to select for successfully transfected cells.

For puromycin selection, use concentrations between 1-10 µg/mL based on the cell type and perform selection for 2-3 days until only transfected cells survive.

 Single-Cell Cloning

Dilution or FACS Sorting: Once the cells are transfected and selected, dilute the cells into 96-well plates to obtain single-cell clones. Alternatively, use FACS to sort GFP-positive cells (if using a fluorescent marker).

Seed the cells at approximately 1 cell per well in a 96-well plate for single-cell isolation.

Allow the clones to grow for 1-2 weeks, replacing the media as needed.

Validation of Knockout

Genomic DNA Extraction

Extract genomic DNA from the single-cell clones using a DNA extraction kit (or traditional phenol-chloroform methods).

PCR and Sanger Sequencing

PCR Amplification: Use gene-specific primers flanking the target site to amplify the region where the Cas9 cut was made.

Run PCR products on a gel to check for indels (insertions or deletions) by size differences.

Send the PCR products for Sanger sequencing to confirm the presence of indels, which are indicative of successful gene knockout.

  T7E1 Assay (Optional)

The T7 Endonuclease I assay detects mismatches in DNA, which are indicative of indels from CRISPR editing. This is done by hybridizing and re-annealing wild-type and mutant sequences, which are then cleaved by T7 Endonuclease I.

Run the digested products on a gel to confirm cleavage, which suggests gene editing.

Western Blot (for Protein Knockout)

To confirm the absence of the target protein, perform a Western blot using specific antibodies against the protein of interest. A reduction or complete absence of the protein suggests a successful knockout at the functional level.

qPCR (for mRNA Knockout)

To confirm loss of gene expression at the transcript level, extract RNA and perform reverse transcription followed by qPCR. The absence or significant reduction in mRNA expression is another indication of successful gene knockout.

Expansion and Further Analysis

Once knockout clones are validated, expand the positive clones for further experiments.

Use flow cytometry, functional assays, or any other downstream analysis depending on the purpose of your knockout study.

 Troubleshooting

  1. Low Transfection Efficiency: Optimize transfection conditions, such as adjusting DNA amounts, using different transfection reagents, or increasing cell density.
  2. No Detected Indels: Ensure that gRNAs target the correct location and that the PAM sequence is correct. Redesign the gRNAs if necessary. Check Cas9 expression using Western blot or qPCR.
  3. Off-Target Effects: Design gRNAs with minimal predicted off-target effects. Use more specific Cas9 variants, like Cas9-HF (high fidelity), if needed.
  4. Cell Death: Knockout of an essential gene might cause cell death. Perform short-term experiments (before cells die) or use conditional Cas9 systems or inducible gRNAs.

 Conclusion

The CRISPR-Cas9 knockout protocol is a powerful tool to generate gene knockouts in a wide range of cell lines. By following the outlined steps—starting with gRNA design, transfection, and selection, followed by validation through PCR, sequencing, and protein assays—you can efficiently create knockout cell lines for research. Proper validation of gene editing, including confirmation of off-target effects and functional loss, is crucial for the success of these experiments.