How to Use Your CRISPR Knockout Plasmid or Virus
A step-by-step guide to enrich for edited cells and detect successful knockout.
Recommended Workflow
Transfect or transduce your cells
Deliver the plasmid using your preferred method: lipofection, electroporation, or viral transduction. Follow your standard protocol for the cell type. Optimize transfection efficiency beforehand if you haven't already — this directly impacts editing yield.
Enrich or select for transfected cells
This step is essential and is the most commonly skipped. Only a fraction of cells will take up the plasmid — if you screen the whole population, signal from unedited cells will mask your results.
Use the selection marker included in the vector:
- GFP marker: Sort GFP-positive cells by FACS 24–72 hours post-transfection. Aim to collect cells in the top 10–20% of GFP expression.
- Antibiotic resistance marker: Begin selection 24–48 hours post-transfection at your validated concentration.
- Puromycin: Typical selection at 1–5 µg/mL; kill curve validation is recommended for each cell line.
Allow editing and recovery time
After enrichment, cells need time to complete NHEJ or HDR and for protein turnover to occur. Residual wild-type protein must be degraded before a functional KO phenotype is detectable. Screening too early is a frequent source of false negatives.
Do not assay during this window — let cells recover, expand, and allow protein levels to fall.
Screen for knockout efficiency
Assess editing at the DNA and/or protein level using methods appropriate to your downstream application. For population-level screens, use the enriched pool. For clonal analysis, single-cell sort into 96-well plates after enrichment, then expand clones before screening. Clonal selection is highly recommended as polyclonal or pooled populations may show residual expression or incomplete editing.
- Genomic confirmation: Sanger sequencing or T7E1/SURVEYOR assay at the target locus
- Protein confirmation: Western blot or immunofluorescence for target protein loss
- Functional readout: Assay relevant to your biological question
Common Pitfalls & How to Avoid Them
⚠ Screening without enrichment or clonal selection
Assaying the bulk transfected population dilutes signal from edited cells. Unedited cells dominate and produce false-negative readouts.
⚠ Screening too early
Residual wild-type protein persists for days after editing. Functional or protein-level assays performed <7 days post-transfection will not reflect true KO status.
⚠ Waiting too long before functional readout
Extended passaging can lead to clonal drift, compensatory upregulation of related genes, or loss of edited cells under certain growth conditions.
⚠ Poor transfection efficiency
If transfection efficiency is <20–30%, even FACS-sorted pools may contain insufficient edited cells for robust analysis.
Typical Timeline Reference
| Timepoint | Action | Notes |
|---|---|---|
| Day 0 | Transfect / transduce | Plate cells at appropriate density 24 h before transfection if using lipofection. |
| Day 1–2 | Assess transfection efficiency | Check GFP signal under fluorescence microscope or by flow cytometry before proceeding. |
| Day 2–3 | Enrich for transfected cells | FACS sort GFP+ population or begin antibiotic selection. Allow 24 h before sorting to ensure peak GFP expression. |
| Day 3–7 | Recovery & expansion | Expand enriched cells. Avoid phenotypic or protein-level assays during this window. |
| Day 7–10 | Genomic & protein validation | Sanger sequencing, T7E1 assay, Western blot, or IF for target protein. |
| Day 10–14+ | Functional assays | Perform clonal selection, confirm editing of isolated clones, then perform downstream biology experiments. For clonal lines, allow additional time for expansion after single-cell sorting. |
Pro Tips
Cryopreserve enriched pools
Freeze down a portion of your enriched, sorted population at Day 3–4 before proceeding. This preserves your work if downstream experiments fail.
Use positive & negative controls
Include a non-targeting guide RNA control alongside your KO construct to distinguish guide-specific effects from delivery or selection artifacts.
Perform clonal selection
Perform clonal selection before validating KO at the genomic level, indel confirmation, or the protein level, Western blot or IF.
Cell-line-specific protocols
Transfection conditions, selection doses, and protein half-lives vary significantly. Pilot experiments in each new cell line are always worthwhile.