Genetic Knock-Down (e.g. with RNAi - siRNA or shRNA) & Knock-Out (e.g. with CRISPR/Cas)

Genetic knock-down versus knock-out and a few different ways to bring it about. If you want to *interfere* with protein production without messing with the gene, you can turn to RNA interference to lessen its protein. But if instead you want to permanently knock the gene out, CRISPR/Cas9 offers you a route.

If you walk into a restaurant at 6am you might get a breakfast menu whereas at 6pm you’d get a dinner one. It’s not that the restaurant doesn’t magically stop knowing how to make a pancake when the clock strikes noon (and if you ask nicely they might still make you one). But they assume people won’t want pancakes at night.

Similarly, your cells produce different proteins at different times but they still have the genetic recipes for making the proteins that aren’t “on the menu” at some point in time - unless you destroy the original recipes, which CRISPR/Cas9 can do, but RNAi can’t. All the original recipes (genes) are written in DNA and locked up in a membrane-bound compartment called the nucleus. To go “on the menu” they have to be transcribed - copied into messenger RNA (mRNA) form and taken into the cytoplasm (general cell area) where the chefs are - it’s this mRNA that RNAi destroys.

As a result, knocking down a gene (such as with RNAi) is like taking something off the menu whereas knocking out a gene (such as with CRISPR/Cas9 genome editing) is destroying the original recipe so the restaurant doesn’t know how to make it anymore. No more pancakes. Ever.

The 2 methods do have other things in common however (In addition to lowering the effects of specific genes). They both achieve their specificity through the use of RNA guides. RNA & DNA letters can base pair with each other - like matching puzzles pieces “A” binds “T” (in DNA) or “U” (in RNA) and “C” binds “G.” So you can get 2 complementary sequences to bind to each other and a sequence can act as a guide to direct silencing machinery to a complementary sequence.

Let’s start with knock-down (via RNAi)…

RNAi relies on small RNA (sRNA) guides. sRNAs are small (only ~22 nucleotides (nt) (RNA letters) long) but mighty! Their sequences “match” sequences in specific mRNAs (protein recipes), so they’re able to bind those mRNAs. And they bring mRNA-destruction machinery with them (before they bind the targets they’re loaded into a protein called Argonaute (Ago) which can shut down some targets on its own and call for backup as needed.

This is a natural mechanism your cells use to take things off the menu (without our intervention). But they use a form of RNAi that uses microRNAs (miRNAs) which are less specific than the ones we use in the lab, but can target multiple related genes (kinda like being able to take all the breakfast-only items off the menu when it’s lunch time).

Our cells use miRNA to regulate over half of all our genes. miRNAs are made from special genes that get processed differently than protein-making genes. And they get loaded into a Ago and guide it to target mRNAs. If the miRNA and target mRNA match completely, Ago can cut it (act as an endonuclease)*. But usually miRNA only match partially - it’s enough to have full complementarity of a 6-8 letter long “seed sequence” - this will keep Ago on there while it calls for backup to help it degrade the target.

*at least our main Ago, Ago2 can - it’s the only** one of the 4 human versions of Ago that can slice
**it was thought this for a long time but recently Ago3 was found to be able to slice in certain circumstances, but using shorter guides

The specific but not *too specific*-ness of miRNA makes it great for targeting related things at the same time, so it’s great for its natural purpose but not great if you want to use RNAi as a tool to artificially knock down specific genes. In that case you want something more specific. You can get higher specificity by introducing guides that are fully complementary so less likely to match multiple things.

And these fully complementary guides also enable Ago to slice (cut) the target, directly harming it, rather than “just” calling in other proteins (cofactors) to help with the destruction.

You can introduce the “guide-to-be” to cells in a dish by transfecting them - sticking in foreign stuff - with double-stranded RNA (dsRNA) that the cells can process into mature guide small interfering RNA (siRNA), or siRNA duplexes themselves. Or you can infect them with a virus (transduce them) that sticks in a circle of DNA called a vector that the cells can transcribe into a short hairpin RNA (shRNA) that can get processed into mature guide. It’s kinda like “give a man a fish” (siRNA duplex) vs. “teach a man to fish” (shRNA) depending on your wants/needs.

Finished in comments

[artsiomshchemialiou]

You couldn't have made this more clear. Amazing, thank you.

[thebumblingbiochemist]

The processing of hairpins and long dsRNA is done similarly to the later processing steps of miRNA precursors, using a protein called Dicer. In the case of miRNA, miRNAs come from genes and get transcribed like protein-coding genes do, but they get processed differently. Their primary miRNA (pri-miRNA) transcript folds up on itself to form a long hairpin (stem-loop) that gets cut into a shorter hairpin by Microprocessor while it’s in the nucleus, then the top of the hairpin gets chopped off by Dicer in the cytoplasm to form a pre-miRNA duplex that gets loaded into Ago, which ejects one strand to reveal the sequence that can bind to target sequences in mRNAs.

Note: Only 1 strand (the guide strand) is needed, but you can’t stick a single strand of RNA into cells or it’ll get chewed up right away. You need to protect it. That’s why we use duplexes or hairpins or long dsRNA instead.

When we transfect those, the effect is temporary because the cell can’t make more of it, it can only use what you give it.

Alternatively, for a more permanent effect, you can stick in a gene vector and tell the cells to make shRNA. It’s kinda like just adding a task to your cell’s normal repertoire - it lives in the nucleus & the cells transcribe it and process it like they would miRNA.

Even in this case, you still aren’t messing with the original DNA gene like you would be with “genetic engineering” tools. You’re just affecting the mRNA copies.

If you want to go mess with that gene, you often turn to…

Knock-Out (via CRISPR/Cas)

This is conventionally done using CRISPR/Cas9, but there are other versions of CRISPR. CRISPR stands for Clustered regularly interspaced short palindromic repeats. it’s a natural bacterial defense mechanism that serves as a bacterial immune system to cleave foreign DNA like that from bacteria-infecting-viruses (bacteriophages) that have infected it in the past.

The original bacterial version uses 2 separate pieces of RNA and a pair of protein scissors called Cas. One piece of RNA has a sequence that matches the target (this sequence comes from a past invader) and the other piece of RNA acts as a “generic” adapter to connect that specific part to the scissors. A key innovation was that you could link together those 2 pieces of RNA into a single piece - a single guide RNA (sgRNA).

Now it can be used as a powerful gene editing tool. You can change the specific guide part to target different genes. But the sequence you target has to be next to a sequence called a Protospacer Adjacent Motif (PAM) that attracts Cas, activates it, and allows for further guide/target binding and cleavage.

CRISPR/Cas9 doesn’t totally annihilate the gene which, remember, is still in the middle of a really long strand of DNA containing other original recipes. Instead, it just makes a cut through both strands - induces a double-stranded break.

The real damage actually comes from when your cells try to fix it. When they do this they often add (insertion) or remove (deletion) an extra DNA letter which shifts the reading frame and turns the recipe into jibberish because the chefs don’t know where to start.

It’s less dangerous to mess with things at the mRNA level (a la RNAi) because those are just copies of the recipe. The original’s safe. And when you “open up another restaurant” by duplicating the cell, that restaurant will have an untouched original copy as well. So off-target silencing is bad, but not catastrophic.

But CRISPR/Cas9 is actually changing the original recipe. So that cell, and any cells that come from it will never be able to put it back on the menu. So you want to be really careful and make sure you’re super specific in your targeting.

So scientists have designed ways to try to guarantee specificity, including mutating Cas so that it only cuts one strand, so you have to target both strands, so 2 separate sequence recognitions are required. And that’s not all scientists have done with CRISPR - there are different Cas-es and you can mutate them so they don’t cut or you can stick in something to put in where you cut - there are really a ton of options.

But because non-bacterial cells don’t naturally have this machinery, you have to get it in there. It’s hard to put in big ole proteins (much easier to transfect siRNA), so scientists have engineered cell lines and animal models to express Cas so that you only have to add the guide.

A couple other differences - since DNA is in the nucleus, but mRNA is in the cytoplasm so the CRISPR/Cas9 action takes place in the nucleus, whereas the RNAi action takes place in the cytoplasm.

So if you were to target the same gene in a cellular restaurant with RNAi or with CRISPR/Cas9 - the end result could look the same - it’s likely that no one in the cellular restaurant would have that food. But in the case of RNAi, the chef might still make it for you if you ask really nice!

In addition to knocking down or knocking out specific genes you know you want to knock down/out, you can use do an “RNAi screen” where you use a “library” of sequences to knockdown lots of different genes in different cells - screen the cells for some effect, then look to see what sequence was in that cell.

Here are a couple papers for reference:



This is an example I show in the video (which was largely the basis for my thesis project in grad school)





    
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