Verifying a CRISPR–Cas9 cut on a DNA gel
Run uncut plasmid and Cas9-digested DNA through an agarose gel: one band becomes two after cleavage.
Goal
Read band positions against a ladder, relate fragment sizes to the Cas9 cut site, and recognise a failed or partial cut.
Apparatus and reagents
Agarose gel, TBE buffer, DNA ladder, plasmid DNA (~5 kb), Cas9–gRNA ribonucleoprotein, DNA stain (e.g. GelRed), power supply, transilluminator.
Procedure
- Load the uncut plasmid sample and note its single band near the well.
- Switch to the Cas9 + gRNA digest and compare: two bands appear because one double-strand break linearises and halves the molecule’s mobility profile.
- Measure each band against the ladder: a 5 kb plasmid cut once should give ~3 kb + ~2 kb if the guide sits 3/5 of the way.
- Look at the NHEJ-repaired lane: indels of a few base pairs barely move the bands — you need sequencing to see them.
What to observe
- Uncut circular plasmid stays high near the well; linearised/cut DNA migrates, and the shorter 2 kb fragment runs ahead of the 3 kb one.
- A ladder lane gives reference sizes; matching band heights to it converts distances into kilobase estimates.
Explanation
Agarose is a mesh: the electric field drags negatively charged DNA toward the anode, and short fragments slip through the pores faster, so migration distance decreases roughly with log₁₀(size). Cas9–gRNA binds the 20 nt protospacer next to a PAM and creates a blunt double-strand break ~3 nt upstream of it; one cut in a 5 kb plasmid yields two fragments whose sizes map the cut site. After cellular NHEJ repair, small indels change the size by only a few base pairs — invisible on a 1% gel, hence sequencing or a T7E1 assay is used to confirm edits.
History of the experiment
Chemists behind it
Related topics
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