Creating Recombination: How Cre-Lox Works
Idea creds @Jellyfish!
Cre-LoxP recombination is a technique used to induce specific mutations; in other words, it's used to edit genes. But it can't edit genes like CRISPR can with whatever sequence you want. (Learn about CRISPR gene editing here.) Instead, it can cause inversions, deletions, or translocations. Below is what each of these look like:
Note: some sources say duplication can also be caused by Cre-LoxP, but I have yet to find an explanatory visual for this phenomenon. So we'll just ignore duplications (another type of mutation) for now lol.
Cre-LoxP recombination uses 2 components: Cre recombinase and LoxP sites. Cre recombinase cuts between LoxP sites using a catalytic triad (just like chymotrypsin!). So, different orientations of LoxP sites will produce different cuts and therefore mutations. While visualizing this, it is important to note that LoxP has a non-palindromic sequence, so the direction it faces affects what DNA region is defined as "between" two LoxP sites.
Below are the orientations of LoxP sites that produce each kind of mutation:
Try to visualize what's going on. In the Inversion, the LoxP sites are pointing towards each other. So, Cre recombinase cuts at both spots inside the area between the sites. This piece flips around and reattaches, resulting in an inversion. In the Deletion, the cut occurs on the right of each one as that's the direction Cre recombinase cuts relative to the LoxP site. So Gene X and the LoxP on the right get cut off together. In the Translocation, the Cre recombinase cuts on the same side in each chromosome and it reattaches as shown. It's really intuitive if you notice that Cre recombinase cuts at the pointy end of the triangle (which represents one specific side of the LoxP site, which I previously mentioned is asymmetrical!), and everything just reattaches.
So why do we use Cre-LoxP recombination? It's advantageous because we can knock out genes selectively, not only in the sense that we can choose which gene to knock out, but also that we can choose which instance of expression of the gene to knock out. For example, imagine a hypothetical Gene Y was expressed in the skin, brain, and liver of an organism. If we wanted to knock out the gene and see its effects ONLY on the brain, then we would have to focus only on the brain. However, most methods of gene editing would also knock out the gene in the skin and liver, which is going to produce extraneous results. To isolate the changes to only the brain, we modify the brain DNA to make sure that's the only place where Cre recombinase is expressed, and also insert LoxP sites in those specific regions. CRISPR doesn't have this localization and often exerts off-target effects, so Cre-LoxP is a good technique for when spatial specificity is needed.
This article was very surface-level, so learn more here:
https://blog.addgene.org/plasmids-101-cre-lox - another pretty simple article.
https://youtu.be/oLPjiwM0G7A?si=zlnu58tLR5sqUQth - detailed, nice visuals, explains further specific applications (like pulse chase experiments, which led to the discovery of Okazaki fragments, except their example concerns stem cells.)
https://en.wikipedia.org/wiki/Catalytic_triad - more on the catalytic triad - good to know for USABO (at least the basics of how it works.)
