CRISPR-Cas9 editing
Cut an early coding exon and let end joining introduce frameshifts. The simplest and most reliable CRISPR experiment; most of the work is choosing a good guide and proving the gene is really gone.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
NHEJ knockout
Cut an early coding exon and let end joining introduce frameshifts. The simplest and most reliable CRISPR experiment; most of the work is choosing a good guide and proving the gene is really gone.
How it differs from the standard method
- Target an exon shared by all transcripts, early in the coding sequence.
- A frameshift in the last exon can escape decay and still make protein.
- Confirm loss at protein level, not only by sequencing.
1A programmable pair of scissors
Cas9 is a bacterial nuclease that carries a short guide RNA. The guide's first 20 bases pair with a matching DNA sequence, and if that sequence is followed by a short motif the enzyme recognises (NGG for the common Cas9 from Streptococcus pyogenes), Cas9 cuts both strands about three bases upstream of it. The guide decides where; the motif, called the PAM, decides whether a site can be targeted at all.
1. Find
20 nucleotides of guide pair with the target; the PAM must sit immediately next to it.
2. Cut
Both strands are cut, leaving a double-strand break the cell must repair.
3. Repair
End joining re-seals the break and often gains or loses a few bases; with a template, the cell can copy an exact sequence instead.
2Two repair routes, two kinds of experiment
Non-homologous end joining is fast, works in any cell, and is slightly sloppy: it leaves small insertions or deletions. Since two thirds of those shift the reading frame, it is the standard way to knock a gene out.
3Measuring what happened
Amplify the locus by PCR from the edited pool. A mismatch-cleavage assay (T7 endonuclease I or Surveyor) melts and re-anneals the product so edited and unedited strands pair up; the enzyme cuts those mismatches, giving extra bands on a gel.
Key terms
Practise these as flashcards →The RNA that targets Cas9: about 20 bases matching the site, fused to the scaffold Cas9 binds.
Protospacer-adjacent motif, NGG for SpCas9. Without it, Cas9 will not cut, however good the guide.
Small insertion or deletion left by end joining; a size that is not a multiple of three shifts the reading frame.
Cas9 protein pre-mixed with its guide, delivered directly; it acts within hours and is degraded quickly.
A site resembling the guide that also gets cut; fewer when the nuclease is short-lived and the guide is unique.
Fitting the mixed sequencing trace of an edited pool (TIDE, ICE) to estimate each indel's frequency.
When to use it
- Knocking out a gene to test what it does, usually by targeting an early, shared coding exon.
- Introducing a specific point mutation, tag or reporter at its natural locus.
- Screening many genes at once with pooled guide libraries.
Compared with alternatives
Changes single bases without cutting both strands, so no indels and no donor template.
Thousands of guides at once to find genes behind a phenotype.
Knocks down the mRNA temporarily rather than changing the gene; useful when a knockout is lethal.
Older nucleases and site-specific recombinases for comparison.
The genotyping PCR across the cut site underpins every readout here.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cut an early coding exon and let end joining introduce frameshifts. The simplest and most reliable CRISPR experiment; most of the work is choosing a good guide and proving the gene is really gone.
- Target an exon shared by all transcripts, early in the coding sequence.
- A frameshift in the last exon can escape decay and still make protein.
- Confirm loss at protein level, not only by sequencing.