Two ways a cell fixes a break
Once Cas9 makes its double-strand break, the cell must close it — a broken chromosome is dangerous. It has two main repair strategies, and which one runs decides the kind of edit you get.
The first is fast and sloppy: non-homologous end joining. The cell simply grabs the two broken ends and stitches them back together. In its hurry it often gains or loses a few letters at the join — a tiny insertion or deletion. The second is slow and careful: homology-directed repair. Here the cell rebuilds the broken region by copying a matching template — normally the sister chromosome, but in editing a template we supply.
Knockout: switching a gene off
Suppose you just want to know what a gene *does*. The simplest experiment is to break it and see what changes. That's a gene knockout, and the messy end-joining route does it for you almost by accident. When the cell adds or drops a letter or two at the cut, it can shift the reading frame — the way the gene is read three letters at a time. A shifted frame, a frameshift mutation, scrambles every codon downstream and usually produces a stop codon early, so no working protein is made. The gene is effectively off: a loss-of-function edit.
Because it relies on the cell's default repair, knockout is the easiest kind of edit and was the first to become routine. You don't even control the exact insertion or deletion — you just need it to disrupt the gene.
Knock-in: writing a chosen sequence
What if you want to *put something specific in* — correct a disease letter, or insert a whole new gene? That's a gene knock-in, and it needs the careful route. Along with the Cas9 and guide, you also deliver a DNA template: a piece that carries your desired sequence flanked by stretches matching the DNA on either side of the cut. The cell, using homology-directed repair, copies your template as it rebuilds — so the change you wrote ends up stitched into the genome.
- Cas9 + guide cut the chromosome at the chosen site, making a double-strand break.
- You also supply a template: desired sequence flanked by matches to both sides of the cut.
- The cell aligns the template to the broken ends and copies across it during repair.
- Result: your exact sequence is now in the genome — a precise knock-in.
Knock-in is far harder than knockout, precisely because the careful repair route is the minority outcome — most cuts get end-joined instead. Raising the share of clean knock-ins is a whole field of effort. The next guide describes a clever way around the problem: tools that never make a full double-strand break at all, and so don't depend on this risky repair lottery.