🎶 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! 🎶 Adaptive immune systems (aka acquired immunity) are 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 some similarities & differences.

CRISPR/Cas stands for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR ASsociated proteins. There are different types, but I’m going to talk about Type II, which is the one people use most in the lab. There are 3 main stages:

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

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. But the PAM sequence is really short & not very specific, so the cell needs to make sure it’s not a “false alarm” – once it 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.

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)

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 (which is why babies can get immunity from their mother’s breast milk)

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. 

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