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Genome editing enters a new phase

Genome editing enters a new phase

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The world’s introduction to genome editing

The world of genome editing is heating up. Since its invention in 2012 of the targeted Cas9 gene editing system, the procedure has garnered massive funding and attention, and it is no wonder, as the potential medical implications are obvious. Quietly in the background, human embryo experiments are already occurring, setting the stage for what might be expected in the near future.

However, the CRISPR/Cas9 system is still not efficient enough for a clinical routine spotlight. That is because the original system does not enact the change directly, but rather, presents conditions that favour DNA change in a desired direction. That does not mean that such an effect will occur, and even novel mistakes can be introduced. This is because the editing system works by introducing a break in the DNA (termed ‘nicking’ in a scientific slang), at a desired location, while providing a pre-programmed template DNA (with the desired change), that cells could then use in the process of fixing the broken (nicked) DNA. The end result is that a cellular repair process introduces the change into the genome. All of that sounds great, except that the cells can resort to another system of repairing the DNA damage by simply fusing together the broken ends of DNA.

Problems arise from the fact that not only is the latter approach used more frequently by cells, but can also result in a random base (single DNA nucleotide in a DNA sequence), insertion or deletion (“indels” for short, and some more useful scientific slang). Therefore we have a system of potentially modifying DNA at a desired location along the genome (giant leap forward from the past approaches), but the biological system does not perform DNA editing directly as it relies on endogenous cellular processes with a questionable efficiency of less than 0.1 to 5%. And it can still get off target!

Image of Merogenomics article quote on CRISPR


New DNA editing kid on the block

That information has changed by now however: already back in 2016, the world was introduced to the first ever Cas9 system that was able to lead to direct DNA nucleotide modification without the need of nicking the DNA. What is fun in recounting this story is how ingenious the development of this new genome editing system was, and how so few in the world have ever heard of it. It also highlights the crazy complexity of science work, which, if executed correctly, can lead to amazing discoveries and progress. The system still depended on guide RNA to bring the editing mechanism to a desired location along the genome (Cas9 uses this short RNA guide to match it with a corresponding sequence along the genome), but the editing mechanism has been outfitted with a novel function.

Image of Merogenomics article quote on gene editing

In a brilliant stroke of foresight, the authors created a fusion between a mutated Cas9 enzyme which could still target the DNA but no longer cut it, and another enzyme, cytidine deaminase. This second enzyme can convert a cytidine DNA base to another one, uridine. Uridine is not part of the DNA code but basically acts in the same way as another nucleotide in the DNA, thymine. In other words, it leads to a conversion of either C to T in the code (or G to A change in the opposite complementary strand of DNA, because C always pairs with G, and T with A nucleotide). So when Cas9 binds the DNA (using guide RNA as a template), it surrounds one of the DNA double helix strands, dislodging the second DNA strand. Thus that dislodged second strand of DNA is in that moment without its counterpart, in a single stranded state. Cytidine deaminase can bind this unwound strand of DNA and chemically convert any cytidine nucleotide to a new base, uridine.

The authors did not waste their time, and showed its effectiveness in 7 different cases that are known to contribute to human disease. But when tested in human cells, the efficiency of base editing was 0.8% to 7.7%, so nothing to write a post about. The authors hypothesized that it was because of the cells’ own corrective mechanisms which removes uridine nucleotides from DNA. Ooops.


Molecular tinkering for molecular good

In order to ensure that they get this top notch publication, they created yet another fusion protein, an even greater chimera with a third protein attached to the system that could act as an inhibitor of cellular uridine removal mechanism. That third fused protein, by the way, was from Bacillus subtilis bacteriophage! Talk about a biological concoction, as this is a virus that invades a main bacteria involved in food poisoning. Bacteriohages, a subtype of viruses, are host-specific viruses and usually infect only specific bacteria species. Aw, truly a marriage till death do us apart. With this new fancy chimeric fusion of a genome editing machinery the efficiency jumped to 20%!

Perhaps these numbers would get the cut for this post, but the authors did not stop there. What they did next was equally ingenious to their prior biological tinkering, as these guys knew their DNA repair background and used it to the fullest advantage. You see, if there are nicks in the DNA in one of the strands, cellular mechanism will remove such damaged DNA and use the other strand as a template to produce a new uncut wholesome double stranded DNA.

If you thought that the authors added yet another (fourth) protein to their fusion complex, then that is just crazy talk! Seriously, who has heard of a four protein fusion? Instead, they reintroduced a mutation in Cas9 so that it would cut only one strand of DNA, the one that is not being modified by cytidine deaminase. This is still in contrast to normal Cas9 which would cut both strands of DNA, so by damaging one selected specific strand of DNA, the authors ensured that the cells would proceed with repair using the strand of DNA with the mutation introduced by the cytidine deaminase.

Image of Merogenomics article quote on Alzheimer

What was the outcome of all of this? Now we are talking about 37% efficiency, and only 1.1% of indel formation frequency. Compared to the original system everyone else would be using, it produced 0.5% efficiency with 4.3% indel formation frequency. That is like a night and day difference!

The authors tested it in neuronal mouse cells that contain the human APOE4 gene variant, which in humans is known to confer increased risk of late-onset Alzheimer disease development. Successful modification of this gene would result in a version that results in lower disease risk. A conversion efficiency of 58–75% was observed with a 4.6–6.1% indel frequency, compared to 0.1–0.3% APOE4 correction efficiency with a normal Cas9 system, and 26–40% indel frequency. Finally, the authors also tested human breast cancer cells to correct a mutant p53 known to be associated with multiple different cancers. Correction in 3.3–7.6% of cells was observed with less than 0.7% indel formation. In contrast, the normal system used resulted in no detectable DNA correction (less than 0.1%), with 6.1–8.0% of introduced indels at the target site. You get the point.

Image of Merogenomics article quote on mutations that cause cancer

This is all exciting news, because the majority of diseases stem from a single nucleotide change in the DNA code. But much work remains before we have a system with pinpoint accuracy that would be desired for routine clinical use. Described here was a method of converting a single nucleotide, where any cytidine is converted in a 5-nucleotide window. Such an approach is still restricted in its scope of possibilities, but we are in good hands here: the lead author of that publication is an expert in this area, and co-founded the Editas Medicine company towards further research in genome editing. I am sure there will be plenty more to hear about from them!

Image of Merogenomics article quote on genetic disorders


Bright future of genome editing

What would be even cooler would be to have a Cas9 system that already possess the catalytic mechanism of cytidine deaminase directly, so that nucleotide conversion could occur on the DNA strand to which Cas9 is already bound. Now that is something to fantasize about! If I am permitted to let loose with scientific fantasies, how about a Cas9-like system that can convert any base of our choosing, where the selection of the base to be incorporated, as well as the timing, and is delivered with Cas9 driven by specific wave frequencies, or some other clever switch button? It’s not as far-fetched as you might think!

Of course, the genome editing machinery is continuously being worked on, not just with its programming to execute the specific editing task, but also in its interaction efficiency with DNA, its delivery to the site, including both entry into the cells, and in finding the correct desired location within the DNA. One good example, that would be interesting to see with the combination of the system as described above, was a set of mutations incorporated into Cas9 that really ensured that Cas9 is nicking the exact DNA sequence it is supposed to. In essence, these mutations help to trap Cas9 in an inactive state if bound to a mismatched DNA sequence.

You can see that this is a very dynamic field of research, and new rounds of clinical trials with genome editing are bound to be around the corner using these newly developed tools. For the rest of us, the best we can do for now is to know in advance what lurks within our genome by sequencing it and cope with using non-genome editing methods. At least Merogenmics Inc can help you with access to the best providers of genome sequencing tests.


This article has been produced by Merogenomics Inc. and edited by Kerri Bryant. 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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