CRISPR origins, from minute to grand

Genetic editing is the ability to alter DNA to produce a desired biological change in a person and it is one of molecular realms that is currently capturing the public’s attention and imagination (for better or worse). More and more people have heard of CRISPR, the acronym for Clustered Regularly Interspaced Short Palindromic Repeats which is the molecular technology of genetic editing hijacked from bacteria and this acronym may soon start competing with “DNA ancestry testing” as being the household name for DNA-related technology.

The above link goes into some detail about the original technology when it was first discovered. For an additional review that has beautiful animations, see the video below.

Since its discovery, a massive amount of research (and money) has gone into CRISPR/Cas9 technology to modify it for the needs of research and clinical use. Just for clarification, CRISPR describes the DNA component that bacteria can utilize to recognize the invading viruses, and Cas9 (for CRISPR associated protein 9) is a tiny bacterial molecular robot that does some important, hard work involved in that process, namely recognizing the right location in the DNA where we want to create a change, and then cutting the DNA so that a change can take place.

We have already written about some of the past advances in CRISPR technology, mainly the mesmerising work spearheaded by Dr. David Liu’s group out of Harvard University. Some examples of these past advances in the quest to alter the CRISPR system to our needs are laid out in this stunning video.

Well, Dr. David Liu’s team is at it again as they recently published yet another astonishing upgrade to the CRISPR system. If you thought their previous invention was wild (which it was), then this one is truly remarkable for its incredible specificity, versatility and low error rate. In the latest and most advanced rendition of CRISPR technology, the molecular machinery can now find a desired target and deliver any type of DNA change desired without the need of introducing double-stranded DNA breaks (breaking the DNA apart essentially) or without using a DNA template to produce the change. The leap forward is simply mind boggling. We are talking about the ability to deliver targeted insertions, deletions, and all 12 possible DNA nucleotide base-to-base conversions. The outcome is so cutting edge that the authors have given it a new name: Prime Editing.

So what advance have they delivered this time?

Newest CRISPR advances: let’s do anything!

Compared to their previous somewhat jumbled ensemble of the past CRISPER/Cas9, this is a much simpler and more ingenious system: a modified Cas9 protein fused to only one more protein - a reverse transcriptase (we will get to that soon).

In the previous system, a piece of “guide” RNA had to be employed to determine the precise location of where the DNA would be cut in half . This cut’s damage to the DNA would initiate a repair process and only then could a DNA template be snuck in nearby in the hopes that during the process of gluing the DNA strands back together, the template DNA would be used by the cell to introduce the precise mutation of interest.

But now in the latest CRISPR, the guide RNA and what would have been a template DNA are transformed into a single RNA entity that authors are calling peg RNA. Part of the peg RNA guides Cas9 to a desired location within the DNA, and another part of the same RNA acts as the template. This RNA template includes some of the same sequence that is already present in the RNA component that acts as a guide, plus the code for mutation(s) to be inserted adjacent to it. While it might sound confusing for now, this is a genius design and we will get to the bottom of it for you!

So vast are the capabilities of this new system that authors purport that it could fix the vast majority of all disease mutations - just wait to find out how many!

All of this new magic of editing DNA at will is executed by two special proteins (molecular robots) that are linked together plus that one RNA molecule that plays two roles in one (peg RNA) but first let us start with the RNA molecule and get into the details.

The RNA molecule is designed in such a way that some of it can be used to identify specific locations within the DNA that we want to modify and at the same time another piece of the RNA provides the code for the specific change to be delivered in the DNA. This is the ingenuous part of the design.

The guiding part of the RNA is used by Cas9 to find the desired location within the massive landscape of the genome. Once found, the DNA is unwound from its double helix configuration to allow complimentary RNA binding. The Cas9 protein then performs another vital task as it cuts only one of the unwound single DNA strands. The original approach required cutting both strands of DNA (which can be dangerous to the DNA’s stability). Cutting only one of the two strands is a does not compromise the DNA integrity as much, as it is much easier problem for the cell to deal with.

The remaining part of RNA that is not binding to the DNA fulfills the role of guiding the DNA change. At first, it extends into the empty cellular space and flips to the side of the machinery complex that is made up of the Cas9 protein and the DNA/RNA duplex. But once the Cas9 cuts the DNA there is also a single strand of DNA extending into empty space, flapping about.

But the RNA portion that is flapping about is specifically designed to be able to recognize and bind to the end of the single DNA strand that was just cut by Cas9. Remember, that loose strand of DNA flapping about is complementary to the DNA strand that was recognised and bound by the RNA portion and used for guiding Cas9 to this specific location.

Once these loose ends of RNA and DNA find each other and interact with one another (a process called annealing like two pieces of magnets coming together) the severed end of DNA can now act as the beginning of a new DNA strand and the rest of the free RNA can act as the template. And within that RNA template you can introduce any mutation you like. So if we are now interacting with that loose strand of DNA, in essence, we are interacting with the two single strands of DNA that used to make up a double helix. And this interaction is done with the same RNA molecule - first in the area of the DNA that tells Cas9 where to bind then it loops around and next it interacts with the other nicked strand of DNA from the unwound double helix. In between these two sites of interaction, in the portion of RNA that had to loop back to bind to the loose flapping end of DNA, is the template for inserting our desired mutation into the future (not yet made!) DNA.

This is where that second piece of protein we mentioned earlier, called reverse transcriptase, comes into action. It has the ability to produce a DNA strand using RNA as a template which does not normally happen in human cells. Instead we have molecular robot proteins that produce a new DNA strand using DNA as a template (they are called polymerases). Reverse transcriptase although alien sounding, is actually a clever name.

The convoluted world of RNA, with a twist of virus

In all our cells we use a DNA master code to produce tiny messages that are RNA. RNA is basically nearly identical to DNA, just slightly different chemically. RNAs can be super versatile in their function! Some of these RNAs, most famously, are the final code to guide the production of proteins - those tiny molecular robots that do nearly all the work in our cells. Some RNAs can interact with proteins (or back with the DNA or even other molecular entities) in a super complex concert of interactions that affect these molecules’ uses inside the cell or their behaviours in the cell and in the process help cells to function to their best ability. Some RNAs even assume complex three-dimensional structures and act as if they were proteins themselves - they become tiny molecular robots in their own right! This is one of the ways that cells can respond to the outside world or change their inner environment.

The sections of DNA that are responsible for generating the RNA that will code for proteins are in fact called genes. And the process of generating RNA molecules from the master DNA code is called transcription. Now you can see the reason behind the name: creating DNA out of RNA template is the reverse of transcription.

Why do reverse transcriptases even exist?

It is viruses that that need them!

Viruses are some simplest biological organisms we know. They are so simple that they are basically only a genetic code in a package. That is it! That is like getting IKEA box with only instructions on how to put your furniture together, but no furniture. When viruses invade bacteria or any other cells, it is the host cell that provides all the machinery to decode the viral genetic information and create more viral particles. Thus in a way, viruses act in a parasitic fashion. The viral genetic code can be DNA, just like that in our cells, but often it is RNA, depending on a virus type.

That means that in viruses that have their genetic code encoded as RNA and not DNA, in order for the invaded cells to make more copies of that viral RNA to create more virus particles, they actually need a DNA template! Remember, our cells duplicate DNA, not RNA so to allow the cell to have access to a DNA template, the virus RNA provides the information on how to create the necessary reverse transcriptase.

Once a virus-invaded host cell creates the reverse transcriptase, this can be used to create DNA versions of the viral RNA and then that DNA version is used to create new amounts of viral RNAs. These are then packaged up in viral envelopes that are also produced using the viral genetic code and the synthesis of many more viral particles is complete. They then burst out of the cell (killing it in the process) by perforating the cell's protective membrane like a Swiss cheese. And the process can then repeat itself with another unsuspecting innocent cell.

As famed Nobelist Alfred Hershey never once said: "Bacterial life is a bitch!"

How to flip a mutant flap

Let’s get back to the new CRISPR technology.

Recall when we last left the scene, peg RNA was binding to a couple of unwound DNA strands of a same stretch of DNA molecule with one of the DNA strands mercilessly nicked by Cas9 protein. Thus our peg RNA was occupied with binding DNA at both of its ends. In between these sites of interaction is the RNA sequence that is the template to create any mutation we want. The reverse transcriptase that is tethered to Cas9 now recognizes the end of the nicked DNA bound to RNA and starts producing a new DNA strand along the available RNA template and voila, we just created mutated version of the DNA.

What happens after the reverse transcriptase completes synthesizing a new DNA strand based on the RNA template? The whole Cas9-RNA complex can fall off the DNA and then the DNA can reanneal itself, meaning, single strands of DNA can pair up again like a microscopic zipper. But… but...

The DNA is faced with an interesting dilemma!

Remember how in the first step we clipped the DNA in one strand? And remember that at the site where that DNA was nicked, we actually extended the DNA from one of the broken ends to introduce a mutation? Sure you do, it is right there in the preceding paragraphs! But did you consider the consequences?!

Well, that means we now have more DNA than we started with! Because we created a mutant version of DNA that never existed before. That means there are two different ways that DNA can anneal itself. Either using the original sequence that was present in the DNA before it was even cut, and if that happens, it will prevent binding of the newly synthesized strand with the mutation (which will just now hang in space). Or alternatively that original sequence can be pushed out by the newly produced sequence (with a mutation) and the original old sequence will hang in the cellular space!

Technically the original sequence should win this contest because there is no mutation present and therefore all nucleotides can happily pair up. Chemically, this is a stronger interaction than for the strand that contains the mutation because the introduced mutant nucleotide(s) will not bond to the opposing nucleotides found in the opposite strand of DNA. Remember when you were in kindergarten and you were asked to pair up with someone and hold hands while your chaperones escorted you about in a tidy neat row? That is what would happen if every nucleotide base finds its complimentary partner to bind to. But there was always some Alex that never wanted to hold hands with Charlie because that was ultra gross and to really demonstrate the indignation for even suggesting that such a pairing should take place, Alex and Charlie would keep as much distance apart from each other as the patience of their guardians would allow, in essence creating an offending bubble in the otherwise neat row of children. That is exactly what happens when two mismatched DNA nucleotides encounter each other that are not meant to chemically pair up.

But the mutated strand has a secret advantage! While the stands of DNA duel for interaction with the complementary (uncut and wholesome) strand, the original strand flap is more likely to be degraded by the cell! As a consequence of the cell's eagerness to degrade the overhanging original DNA strand (based on how the broken/nicked end of DNA is recognized by the cell's clean-up proteins), the mutation is favored for incorporation into the DNA! The authors coolly call this process "the flap resolution". You don't hear that everyday!

Overall, ingenious but a really simple and smart process too, right?!

But don’t forget it was the culmination of decades of work by thousands of scientists to unravel many secrets of biology that had to happen to be able to manipulate nature into an intricate and novel design such as this one!

Now, what about the issue of CRISPR introducing mutations into other, undesired locations of the genome (referred to as off-targeting)? That has been the reoccurring issue with current CRISPR technologies and the curse responsible for reducing its clinical potential.

Here too the new Prime Editing CRISPR shines in comparison. Compared to treatments for specific genetic editing that have previously been shown to cause off-target mutagenesis with typical CRISPR approach, this new approach resulted in only <0.1% off-target issues - dramatically lower than the traditional approach (4-48% depending human cell type used). One notoriously difficult cell line showed 2.2% off-target issues with the new CRISPR compared to 60% with the original approach!

When testing for correction of real life genetic diseases in a variety of cell lines, the efficiency of correction ranged from 20-58% (number of cells successfully targeted by the system) and introduction of off-target mutations was minimal and ranged from 0.13-4.8%, depending on cell type.That is compared to 1% correction efficiency of the original CRISPR system!

After developing such a remarkable technology that is so versatile in its ability to correct or deal with so many different genetic issues, the authors perhaps understated this remarkable conclusion: "Because 85–99% of insertions, deletions [...], and duplications in ClinVar are 30 bp in length or smaller, in principle prime editing could correct up to about 89% of the 75,122 pathogenic human genetic variants in ClinVar." ClinVar is the public database that connects information related to the clinical impact of DNA mutations. That is a lot of pathogenic genetic information already! We loved the figure the authors provided showing what type of mutations drive the human disease development so much that we thought it was worth showing here as well.

Adapted from Anzalone AV et al. 2019. Nature doi: 10.1038/s41586-019-1711-4

This means we now may be at a point of being able to tackle correcting the vast majority of human diseases! In theory, we can now drive the evolution in any way we want!

In theory.

In the meantime, international scandals related to misuse of genetic editing technology have already begun and we are certain they will continue but we are also certain that the main driving force of genetic editing technologies will remain the eradication of human suffering. But when the authors of the Prime Editing mechanism mention that 89% of all human disease via DNA mutations could be corrected, what is meant by that? Ideally you would want disease to never materialize in an individual - but that would mean more designer babies!

For now, genetic editing technology remains available to a scant few, as it is in its infancy and has only just begun to be examined in post-birth genetic conditions. But the technology makes leaps and bounds increasingly and surely your strongest bet is to get ahead of the curve and take control of your genetic information by having your personal genome sequenced. By taking this step now, you begin by being able to cross-reference with the ClinVar and other medically relevant genome databases. This is only the beginning and down the road it is entirely feasible that the problematic mutations discovered in your personal genome sequence will be dealt with by using specialized genetic editing techniques which will minimize the onset of disease symptoms. Looking further into the future, once the field matures and Genetic Editing becomes commonplace with safety guidelines and full ethics approval, perhaps genetic editing will be available prior to birth (or fertilization) to ensure the birth of a genetically healthy baby.

We can hardly wait for the next exciting iteration of Genetic Editing technology and hope it can start delivering on the incredible potential soon!!

This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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