The headline skill of modern biology, at your own bench. You'll direct Cas9 to a single spot in the E. coli genome and rewrite one letter of its DNA — a change the cell then carries for life.
CRISPR-Cas9 is a programmable "find and cut" tool. A short guide RNA (gRNA) tells the Cas9 protein exactly where to cut the genome. When the cell repairs that cut using a template DNA you supply, it copies in your intended change. In this classic experiment, a single base change in the rpsL gene makes E. coli resistant to the antibiotic streptomycin — so edited cells survive on streptomycin plates and unedited ones don't.
On ice, add the Cas9/gRNA and the template DNA to your competent cells exactly as your kit specifies. Swirl gently; keep everything cold.
Incubate on ice ~20–30 minutes so the DNA associates with the membrane, just as in a standard transformation.
42 °C for 45 seconds, then straight back to ice for 2 minutes. This drives the CRISPR machinery and template into the cells.
Add recovery medium and incubate at 37 °C for ~1–2 hours. Cas9 finds and cuts rpsL; the cell repairs the cut using your template, writing in the resistance mutation. Cells also need this time to express the edited gene.
Spread onto streptomycin plates — only cells that were correctly edited survive. Spread a little onto plain LB too, to confirm your cells are healthy.
Invert; incubate at 37 °C for 16–24 hours. Colonies on streptomycin are your successful edits. Compare counts to your controls to judge editing efficiency.
| You see… | Likely cause | Fix |
|---|---|---|
| No colonies on streptomycin | Low editing efficiency or failed uptake | Verify heat-shock timing. Keep cells cold. Extend recovery. Confirm you added the components in the right order. |
| Nothing grows anywhere, even plain LB | Cells died | They warmed up or the antibiotic contaminated everything — use fresh competent cells and clean plates. |
| Lawn on streptomycin | Spontaneous resistance / too many cells | Expected at low frequency. Plate fewer cells and compare against an unedited control to see the real effect. |
| Unexpected colony types | Contamination | Return to sterile technique; remake selective plates. |
Cas9 is an enzyme guided by a 20-letter RNA sequence that matches your target in rpsL. Where the gRNA base-pairs with the genome (next to a short "PAM" motif), Cas9 makes a double-strand cut. A cut chromosome is lethal unless repaired — so the cell uses the template you provided, which carries the desired single-base change flanked by matching sequence, to patch the break by homology-directed repair. The repaired gene now encodes a slightly different ribosomal protein that streptomycin can no longer disrupt. The antibiotic plate then selects for exactly those cells.
The Bacterial CRISPR Editing Kit ships with Cas9, the guide RNA, template DNA, cells, and selective plates — everything in this protocol.