When you hear about gene editing in the news you may understandably get confused. But after this post there’s no need to whisper, “What’s the deal with that thing called CRISPR?” Here’s your Insta-Class on CRISPR/Cas!
Proteins are like cellular machines with lots of working parts (or at least hopefully working parts). Genes hold the instructions for making proteins, so if you change the gene (GENETIC ENGINEERING aka GENE EDITING) you can change the protein, and if you totally mess up the gene, you can prevent the protein it codes for from being made all together. And scientists can take advantage of these relationships in order to see how proteins work and what they do – and even to treat diseases – using variants of a system called CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR ASsociated proteins). Scientists and doctors have only recently started harnessing CRISPR’s power – but bacteria have known about it for years!
CRISPR/Cas is a way to use RNA as a guide to direct a protein called Cas (Crispr-associated-protein) to a specific location on DNA (target sequence) and cut it. Bacteria have it naturally – they use it as an immune system – they’re interested in cutting up DNA from invaders like bacteriophages (bacteria-infecting viruses) so they make guide RNA that matches sequences from a running tally of invaders they’ve encountered before. If the invader tries it again, it can be recognized as foreign and cut.
Scientists interested in editing DNA (genetic engineering) have harnessed its power and “flipped it around” – introducing foreign RNA “guides” to cut “non-foreign” DNA in all sorts of cells – and to edit it. Because, you see, cells don’t like having their DNA cut (not that you can really blame them…) so when DNA gets cut, cells will try to fix it. If you give it a matching piece you can have the cells put it in when they fix it, and thereby change the sequence. If you don’t give it an insert, it’ll try to stitch it together but often makes mistakes that make the cell not make the protein, so you can use it to “knock out” genes
But how did this tool for knocking out itself come about? Like many tools biochemists and molecular biologists use, CRISPR/Cas comes from the itty bitties – microorganisms like bacteria and archaea (another type of single-celled living thing). Because these creatures only have 1 cell, they can’t use a complex immune system like we rely on, where immune functions are relegated out to other specialized cells. Instead a single cell has to be able to do it all – including the ability to “recall” invaders it’s seen before they get the cell to fall.
The ability to remember past invaders and be “on alert” for them is called adaptive immunity (aka acquired immunity). It’s how organisms “learn” from infections so that they can recognize & respond to them more effectively if the invader tries again. Our adaptive immune systems rely on antibodies, which are little proteins that recognize parts of foreign proteins by their “shape.” Bacterial immune systems rely on CRISPR RNAs (crRNAs), which are pieces of RNA that recognize parts of foreign DNA by their sequence.
Both systems rely on “unique parts” that recognize specific invaders and “generic parts” that allow the unique parts to be processed & displayed. In our adaptive immune systems, the unique parts are the variable regions of antibodies, and they come from “trial and error” – randomly producing antibodies and then making more of the one(s) that, by chance, match.
Instead of this random but effective approach, bacterial adaptive immune systems go straight to the source – they take a bit of the invader (often bacteria-infecting viruses called phages)’s DNA and use that as the unique part. So it, by design, will bind the invader if it tries again – if it survives that initial attack…
Let’s look a little closer at how CRISPR works & some similarities & differences to our immune system. There are different types of CRISPR (it has had years and years to evolve differently in different itty bitties), but I’m going to talk about Type II, which is the one people use most in the lab. There are 3 main stages to how it works:
ADAPTATION (aka spacer acquisition, aka immunization): bacteria insert pieces of foreign DNA as “spacers” between repeated sequences in their own genome (collection of DNA) in a CRISPR ARRAY, which serves a “running tally” of past invaders 🎶 CRISPR’s got a long list of ex-invaders, they’ll tell you Cas9 cut their DNA. But CRISPR’s got a blank space, baby, & phage, it’ll write your name! 🎶
EXPRESSION: All the components of the CRISPR/Cas system are expressed as an OPERON – this means that it’s all expressed together as a “package deal.” So, at the same time the guide RNAs are made, so are the necessary proteins (Cas proteins) & adapters (trans-activating CRISPR RNA (trcrRNA))
CRISPR arrays are transcribed (an RNA copy made) from a promoter with a leader sequence into one long pre-crRNA and processed into individual mature crRNAs; Cas proteins are transcribed into messenger RNA (mRNA) that’s then translated into protein. RNA parts (tracrRNA & crRNA) + Cas effector nuclease (Cas9) = active surveillance complex with its eye out for the invader that has a matching sequence (protospacer)
INTERFERENCE: If the invader tries again, this surveillance complex will find it, bind in, unwind it, cut it & degrade it.
Some more details:
ADAPTATION PHASE – Cas9 is the protein that cuts the target in the interference stage, but there are other Cas proteins needed to cut the target out of its original home (invader’s genome), cut the array open at a repeat, and stick it into the array for the adaptation stage. Cas1 & Cas2 help with this part.
a couple important notes here: the repeat is cut staggerdly & the overhangs get filled in so the new spacer gets inserted without having to erase any existing one.
the part of the target that complements the spacer is called the “protospacer” and, in the target, it’s next to a short “code word” called a PAM (Protospacer Adjacent Motif) (in SpyCas9 (Streptococcus pyogenes’ version) this is just NGG where N can be any letter). The PAM is NOT inserted into the CRISPR array (is not part of the spacer) & this prevents the cell from attacking itself as we’ll see…
EXPRESSION PHASE – crRNA maturation: pre-crRNA has to get processed into mature cRNAs – the individual guides get separated. This is part of the reason you need those generic repeats – they’re like the “dotted lines” that tell the cell where to cut them apart. They match part of the sequence of an “adapter” RNA called trcrRNA. This makes a section of double-stranded RNA (dsRNA) that another pair of scissors called RNase III recognizes & cuts.
The repeats also provide a way to connect them to the Cas9 protein, which will cut the foreign DNA – part of tracrRNA recognizes the Cas protein & part of it recognizes the generic repeat part of the guide RNA
INTERFERENCE PHASE: When it goes on the hunt, Cas roams around bouncing off of things until it lands on a piece of DNA with a PAM sequence (protospacer adjacent motif). This is the first signal that something might be “foreign” Different bacteria have slightly different Cas-es which like different PAMs, and they keep that PAM away from the spacer copy that’s in their own genome, so they don’t confuse self (version in array) for foreign.
So if they recognize that sequence a red flag goes up. CRISPR/Cas’ version of a SPAM filter is a “PAM” filter! But the PAM sequence is really short & not very specific (unlike the part of the guide that has to match the target (the SPACER), which is like 20 letters long, the PAM is simple – just a few letters. For the “classic” Cas – SpyCas9 (“Spy” because it’s from S. pyogenes not cuz it’s spying on invaders…) the PAM is just “NGG”. The N is just shorthand for the “any letter”).
This short-but-sweetness of the PAM means it occurs by chance relatively frequently, so CRISPR can target genes all over the place – but it also means the cell needs to make sure it’s not a “false alarm”!
The DNA lets in the PAM interact with specific protein letters (amino acids) in Cas. So Cas kinda roams around the Cell colliding into things and if it lands on DNA where there’s the right PAM it will get stuck momentarily. And this momentariness is enough for it to shape-shift a bit and disrupt the double-strandedness of the DNA, giving the guide the chance to start sneaking in…
Once Cas binds to the PAM it starts to unzip the DNA & look around. If the guide matches it’ll sneak its way in between the DNA strands, peeling away one strand so it can partner with the other. But this is in the middle of a big ole chain, so instead of the second strand falling off, it just bulges out in something we call an R-loop -> RNA bound to DNA and the other DNA strand bulging out.
Now Cas really knows something is foreign. So it cuts the DNA (the binding and unwinding also positions the DNA in the path of Cas’ 2 pairs of scissors – the HNH motif & the RuvC motif. It does this cleavage 3bp upstream of PAM.
If you design a guide that matches something you want cut (and is next to a PAM), you can get Cas to cut where you want. If you provide the cell with something to insert that has parts that match the part you broke, it can fix the break using homology-directed repair, putting in the insert.
But If you don’t give it anything else, the cell will just try to fix it the best it can using “non-homologous end joining.” Since the cut is blunt (straight across) you don’t have “sticky ends” like you’d get if you used restriction enzymes. So your cells just want to glue the strands back together before they swim apart because you won’t know how to put it back together. So it kinda rushes to fix it in a process called non-homologous end joining (NHEJ) which makes a lot of mistakes
And these mistakes can be “fatal” for the protein that the gene’s supposed to make, effectively “knocking out” that gene so the cell can never make it again. So it’s a powerful gene editing tool for scientists who want to study what genes do – knock it out and see what happens in its absence. What can the cells “not do”?
When scientists want to use CRISPR as a tool, they have to introduce all the components because our cells don’t use CRISPR naturally – instead we adopted a different adaptive immune system, based on proteins called antibodies or immunoglobulins that recognize foreign things (antigens) and mount an immune response. An antigen is anything that triggers an immune response (even something “harmless” like a peanut) whereas a pathogen is a disease-causing microorganism like a virus or a bacterium)
Instead of that “take notes” approach of bacteria, our adaptive immune systems work by a sort of trial & error approach – immune cells recombine DNA sequences to make lots of different antibodies. Most of these won’t work, but, when one does, it gets added to your body’s “permanent collection” – the cell that has that “winning lotto ticket” specializes in making it and it makes more copies of itself. This puts the pathogen on the immune system’s “watch list.” Now, if that same pathogen tries to invade you, you’ll have antibodies that recognize it as foreign and call in the molecular assassins
A bit more detail: the “experimenters” are progenitor B-cells. They’re like “blank slates” – gene rearrangement occurs (they mix & match different genetic “options” for the antibody’s final form) & they start making a unique antibody receptor that sticks out from its membrane. Now it’s a MATURE B CELL. But it’s still “naive” – it hasn’t encountered its matching antigen.
If it does, it’ll DIFFERENTIATE into 2 kinds of B cells – EFFECTOR B CELLS (aka plasma cells) & MEMORY B CELLS. Effector B cells make lots of that antibody (each can make millions of the antibody molecule) &, instead of displaying them on their surface, they secrete them into the bloodstream for a wider-reaching response
The memory B cells display the antibody on the surface like the original naive cell, but now in the “permanent collection.” Unlike with CRISPR, this “permanent” memory isn’t stored in our genome, and it isn’t passed down in our genes. Only that subset of cells knows it. Those memory B cells live in the bone marrow (which is why bone marrow transplants “swap” someone’s immune memory) & can circulate between lymph nodes – they secrete a low level of antibodies to keep watch.
So if the body encounters that invader again, it doesn’t have to search through billions of potential antibodies to find a match – it has one in the “permanent” collection, it just has to make more of it.
Scientists can take advantage of both these types of immune systems. We can harness the power of CRISPR for gene editing, and we can use antibodies to check to see if certain proteins are present in all sorts of experimental scenarios. A common use for antibodies in the lab is Western Blots, where you separate proteins by size by running them through an SDS-PAGE gel, then transfer the proteins to a membrane & use antibodies specific to different proteins as a probe to see if they’re present. http://bit.ly/2Iy5b6r