#most of my research is on rna and protein expression and not dna modification

[ladysqueakinpip]

the only thing my sister and i have to offer to the rise fandom is theories about how lou jitsu's dna recombined with the turtles

[philosophicalparadox]

Demon Splicing and Why the Clones Failed.

This is more like a research paper than a fan theory, but hey, I had fun. I’ll try to improv some citations at the bottom, but I don’t guarantee that Tumblr will let me keep them there. 

Now, this is a HUGE, MASSIVE, LONG-ASS POST, and once more it is ultra-sciencey; so if you have any confusions, questions, or want to just look the other way and just go “eh, magic” then that is totally okay. I don’t, and won’t, claim to be any kind of authority on these things; I just needed an excuse to open my computer again and I guess do some intense research into etymology (that would be the study of anything with an exoskeleton, basically. I promise it is very much relevant to this theory.). 

Summary/TLDR: Demons need specific qualities in their hosts in order to suit themselves, ergo they modify their host’s bodies by using Mutually symbiotic viruses, akin to those of the Polydnaviridae ingroup which coexists within several genera of parasitoid wasps, to alter the human genome. What we explore here is how they do so. Also, because it is intrinsically connected, we will also be dipping our toes into why, exactly, the clones weren’t “suitable” in all instances, as well as how demons may or may not select their hosts. This circles back to my previous post discussing the Twin’s and Paternity, and specifically the topic of genetic expression, though you do not need to have seen or read that post to understand what I’m talking about here. Also discussed is the matter of genes that humans lack, but which would seem to find their way in during possession; the production of feathers, the formation of additional limbs, proteins,  and such which are simply not within the power of any existing virus we know of to alter . 

One thing must lead to another however, so before we get into the biological science, we need to get into the hypothetical, cosmological stuff that is quantum physics. Didn’t see that one coming, did ya?

Demons and DNA: 

We know from that one elusive panel of chapter 44 ( I think...) that demons have genetics - they have genes, which implies that they have, at the very least, DNA. The question then is how, but more so where - where did those genes come from?. Demons don’t have physical bodies, right...? Why would they need DNA? 

Because maybe some of them do possess actual, physical “bodies”, or at least cells, that preside in Gehenna. 

The Demon Kings are quite likely to be an exception rather than a rule, considering they were the first demons to have come into existence, or at the very least the first demons to have ever attained bodies -- which is precisely how demonkind may have obtained DNA in the first place, via a phenomena called horizontal transference. 

Now, I’m going to contradict, in a sense, my other post here, and tell you to forget what you were taught about viruses in high school. Virology is a complicated school of biology, and viruses are extremely simple, and yet extremely complex organisms. Now, viruses typically contain RNA which allows the virus to reproduce once it is injected into the cells of its host by combining viral RNA with eukaryotic (for the sake of simplicity) DNA. 

However, there are strains of viruses that contain DNA, not RNA. No one is completely sure how these viruses evolved, but one theory would suggest that these dnaviruses “stole” part of their genetic material from the hosts they evolved with, incorporating pieces of lipids and proteins to turn their RNA into functional DNA; this process of one organism “stealing” DNA from another is called horizontal transference, and it is how bacteria and other asexually reproducing organisms maintain genetic diversity and “evolve”. 

But, you ask, how the bloody hell does a Virus have DNA? How does it replicate?

 When most people think of viruses, they think of mobile ones, pathogenic ones - but dnaviruses are not usually pathogenic, instead highjacking the excretory or reproductive systems of their hosts and using their reproductive cells to spread genealogically from parent to offspring. One well-studied example of this is the polydnavirus found in Ichneumon wasps, which are themselves parasitoid. They reproduce by injecting their eggs into the bodies of paralyzed caterpillars, who then feed the hatching larvae with it’s living tissues. However, one problem the wasp faces with this method of reproduction is the caterpillar’s immune system, which could kill the eggs - were it not for the polydnavirus, which produces chemical signals that prevent the caterpillar’s immune system from destroying the precious egg that is it’s host cell. As the larvae develops, the polydnavirus is replicated into the cells of the larvae, and once it hatches it is literally born with the virus in it’s body. (I’ll let you go wild with the half-demon thing there, I’m here to talk about possession right at the moment.)

Ok, ok, but what does this have to do with demons? after all, demon possession is, in a way, “contagious” since demons can go from host to host. 

Welcome then, to the world of multi-viral mutual symbiosis - fancy way of saying viruses can work together to meet the ends of one another in a host if it benefits both viruses. Demons may possess some form of this event, being somehow sentient (by means perhaps of primitive, conductive cells not unlike what you would find in a jellyfish) but ultimately composed of or utilizing not only one, but several strains of viruses to fulfil their parasitic ends, one which allows them to infect the host and modify existing DNA, and one which can incorporate it’s own DNA into that of the host to bring about desirable conditions. To that, I must add as a courtesy that those primitive conductive cells which could, in a way, offer sentience, may in fact be what comprises the physical manifestations of demon’s hearts. None of this is, of course, to explain demon magic, which is a subject I do intend to breach one of these days - but not today. Today, we do science.

 This goes away to explain why Todou sprouted feathers, a phenomena that would not have otherwise been biologically possible given the constraints of human protein structure. That isn’t to say that it would be impossible for a virus to modify via RNA transcription keratinoid proteins to form hollow attachments, which is exactly what you find in polar bears and porcupines, but the structure of feathers is, I’m afraid, just too far off the mammalian path for it to be but a 0.03% likelihood via RNA transcription alone, meaning that it would have to have been the result of DNA that isn’t human. 

Speaking of statistical probabilities: 

Cloning and the Failure Thereof

Humanity has a hollywood-induced idea that cloning organisms is a fail-less system, when that could not be further from the truth. In point of fact, only about 3% of all attempted cloning experiments with everything from fish to sheep produce viable, healthy clones. This is because cloning is done, kind of ironically, in much the same way as a virus operates; by using the  DNA and RNA of the existing mother’s cell’s to complete the chromosomal pairing up that normally happens in the zygote during fertilization. Because of this, the RNA transcribes, ideally, the same exact DNA code that the “mother” has; but here again we get into genetic expression, because though a clone is genetically the same as it’s parent, it is exactly BECAUSE it is genetically identical that recessive (and often in the case of  some experimental animals, fatal) traits and gene combinations can occur, depending on exactly how the original, zygotic DNA is copied. Even when using the RNA of the same organism, transcription errors naturally occur -- and they occur so frequently, in fact, that very few cloning attempts are ever successful; that is, they either produce genetically weak, fatal-combination, infertile, or underdeveloped offspring that ultimately can’t be re-cloned or which can not reproduce, and therefore negate the incentive to clone an organism for it’s “healthy genes”. 

Connecting the dots: 

When a demon is cloned, it’s human DNA is cloned; but so are the genetic modifications of the dnavirus, which is why clones seem to have human superpowers. They are no loner 100% genetically human, and that opens the door to all kinds of genetic complications and probably meant that thousands, not hundreds, of clones were “discarded”, and hundreds died before they even lived. Simply put, it’s an absolute bloody miracle that the cloning thing worked at all, much less that Lucifer was able to remotely perfect the technique. 

How he did so is not so much a mystery though; unlike what you would assume, with mammals at least, the more often you re-clone a clone, the “cleaner” it’s genetic code seems to become by phenomena of natural selection and artificial selection; clones with good genes are re-cloned, clones exhibiting bad genes are culled or die on their own, and so on and so on until you get a good sized population of identical clones. With the added fuel of the elixir to make growth happen phenomenally fast, it’s not too surprising that he has a private stock of cloned bodies to inhabit whenever he likes. (Which gave me big Orochimaru vibes, just sayin’). 

As for the RNA virus body, I suspect that is retained with the demon at all times, which makes sense because once and RNA virus stops replicating it’s RNA into the host, the host cells re-fix the “broken” codes and eventually replaces the alien DNA created by the virus with it’s own; however, a dnavirus’ DNA gets worked semi-permanently into the system of it’s host, since it has it’s own completed code which is then, reversedly, transcribed over and over by the host’s RNA transcription, which is why dnaviruses went undetected by science until about 20 years ago, and why, God forbid, if there was ever a pathogenic dnavirus, we would all be royally screwed because even the best immune system on earth can’t detect a dnavirus because our immune systems rely on identification markers dependent on RNA viruses; oddly, however, so does every other organism, meaning there literally is not a single living thing, including caterpillars and spiders who are victims directly of “pathogenic” polydnaviruses, has an immune system that could find the damn things. They utilize the host’s own RNA to transcribe their DNA, and therefore go almost completely undetected by whatever they infect.  

Speaking of which, let’s talk about:

Immunity and Prions

If Demons rely on RNA viruses to primarily infect their host, then it would make sense why some people would be more resistant than others; however, there  is a compelling aspect of demon possession which makes me think that it is the other way around - everyone is resistant, until they are not. 

Demons typically possess bodies which have weak-minded and psychologically stressed individuals behind them. Stress weakens the immune system, but it does so in specific ways; and certain viruses in real life are programmed to take advantage of these specific measures more than others. 

Right now in the US, there is a nasty epidemic of CWD, Chronic Wasting Disease, spreading through native deer populations on the east coast. This “zombie disease” is a virus that infects the nervous system of the deer (along with cattle and sheep) and forms prions - folded proteins that are then replicated, and replicated, and replicated; and like cancer of the brain, they just keep on replicating and replicating, eating up the animal’s energy reserves and drastically impacting their behavior and bodily functions, starting by supressing and outright destroying their immune system. Mad Cow Disease is a more famous example of a prion disease in the same family as CWD, except that those prions migrate; they move into the soft tissues of the animal and make every single part of it impossible to eat without also contracting the prion, which contains the virus; and MCD is not remotely picky about it’s host, since it affects a very basic protein structure. Any and everything from birds to reptiles to humans can be infected by MCD and it is completely fatal. 

My point is, that CWD and MCD both primarily infect animals exhibiting high levels of stress hormones, which is why outbreaks happen primarily during the breeding seasons for these animals. Not only that, but the virus then directly attacks the animal’s immune systems and opens them up to every kind of secondary infection you can imagine. 

However, prion diseases and even just plain old viruses can do the exact opposite as well. HIV is a common virus that kills you by making your immune system hyperresponsive, not by shutting it down; it becomes so responsive, in fact, that it attacks healthy tissues. Prion diseases which affect insects also do this, creating folded proteins in the nervous system of the bug that trigger it’s immune system to continuously flood the body with antibodies until it is just too exhausted to do so, and the insect’s body decays as a result of secondary infection. 

It could be that this is the case of demons as well. Prions would be valuable in affecting the behavior of the host, though not necessary; they would, however, make the ingestion of a possessed person almost guaranteed to infect you, since most viruses just don’t have the defenses on their own to tackle stomach acid, but a prion virus does. 

To recap: 

Demons use DNA and RNA viruses to infect and modify their host to their liking, perhaps using the assistance of prions to aid in endurance and transmissibility. Because of this, cloning is a gamble of “what DNA will I pull out of the box today” since the DNA virus’ DNA, and possibly even any prions, is left behind even after the parasitic demon leaves; however, the RNA virus is inert once it leaves a host body, and therefore is retained by the demon within whatever primitive cells they may carry in their demon hearts, which may be taken from some immutable “form” or body that they possess on the other side of the divide (in Gehenna); these alien forms may be the byproduct of their first ever possession, using, perhaps, horizontal transference to absorb some of the DNA from their first (and possibly even subsequent) host and then re-incorporate it into subsequent hosts, which is how Amaimon would be reptilian in spite of having a mammal body; because he perhaps, first possessed or found genetic favor of a reptile of some kind and “borrowed” the DNA from them via horizontal transference, since it worked for him. This can then be applied in turn to all other demons, or at least demon kings. 

DISCLAIMER:

I spent literally a week researching this stuff, but I am welcome to criticism of my shoddy work. Also, I am in no way saying this is technically right; it’s just a theory after all, and you’re more than welcome to disagree. :)

If anyone wants to add on, feel free. :) I think I’m done for the week. 

[just-be-excellent]

CRISPR - Cas9

Never done this before, but I have a bunch of essays (30+) I wrote during my revision for finals this year. I don’t have any references for the reading I did for these essays, but they are largely based on my lectures - please forgive me, fellow scientists. Also please bear in mind that this was written for revision purposes at an undergraduate level and may lack detail or contain errors.

I find this super interesting. So should you. 

The CRISPR-Cas system was first identified in bacteria as a form of immunity against phages. Bacteria acquire parts of DNA that correspond to phage DNA, and it is inserted into the CRISPR locus. This locus contains many segments of DNA that correspond to the species history of infection. The CRISPR locus is transcribed and processed into crRNA, which are then incorporated (along with tracrRNA) into a Cas protein for targeting to the corresponding phage. This prevents replication of the phage genome and infection.

It was first characterised in 2002, when it was noted that many bacteria have repetitive sequences close to the same family of proteins (Cas proteins). 5 years later, in 2007, it was identified that these clusters of repeats corresponded to known parts of phage genomes. It was then hypothesised that these were involved in phage resistance. This hypothesis was tested experimentally, by the selective removal of certain CRISPR loci. The result was a loss of immunity against the corresponding phage, confirming the initial hypothesis. Also tested was the requirement for the Cas proteins, which were also seen to be required for resistance, as their removal led to a complete loss of immunity.

Several types of CRISPR systems were then identified, including types I, double strandedII, III and U. Type II is the most relevant to genome editing as it involves only one Cas protein, Cas9, to incorporate guideRNA for targeting to the sequence.

Two RNAs were identified to be crucial for the CRISPR-Cas system, tracrRNA and crRNA, both being required to guide Cas9 to the target sequence. crRNA is synthesised from the CRISPR locus, with tracrRNA transcribed from the same promoter in the opposite direction. These two RNAs hybridise together to give a double-stranded molecule, with part of the crRNA overhanging at its 5’ end. This overhang is the section of RNA that hybridises to the target DNA and guides the Cas protein, resulting in a double strand break from the endonuclease activity of Cas9.

Once the target sequence has been cut, there are two possible routes of repair for the cell. Homology-directed repair is the least error-prone of the two, however it requires a homologues piece of DNA to be present (normally the second chromosome of the pair). Alternatively, non-homologous end joining is also possible, with the two ends being directly ligated together. This error-prone repair mechanism can result in small insertions or deletions of sequence, resulting in a frameshift of the gene.

In 2014 there was a breakthrough in CRISPR-Cas9 research, which discovered that the crRNA had a short 20nt region that hybridises to the DNA. This sequence can therefore be edited to any sequence, to program Cas9 to target any gene, in any cell, in any organism. 

Before this breakthrough, RNAi was the state of the art method for studying protein function in mammalian cells. This was first discovered in the 1990’s in plants, but it wasn’t until the early 2000’s that its application in mammalian cells was realised. siRNAs are incorporated into the RISC complex to target the complex to mRNA for degradation. This results in the knockdown of protein expression in cells, however trace amount of the protein will remain. This technique had shortcomings in other areas also, as the results were not stable or inherited.

CRISPR-Cas9 offered a potential technology that could knock out genes fully, or insert/engineer genes with relative ease in any organism. Better yet, the manipulations were stable.

Further research allowed the production of an engineered chimeric RNA that combined the features of both tracrRNA and crRNA. This meant there were fewer components to the system, and therefore reduced the complexity – now requiring modification of only a 20nt region to target any gene (however the cleavage site must be directly followed by an NGG motif, alternatives that do not require this are being investigated).

Following the double strand break produced by Cas9 and the chimeric guideRNA, the two repair mechanisms can be exploited to manipulate the genome is two distinct ways. Non-homologous end joining can be used to produce stable gene knockouts via frameshifts resulting from small insertions/deletions. Alternatively, homology-directed repair can be exploited to insert whole new genes. Instead of the second chromosome of the pair being used as a template, a plasmid can be introduced that contains the gene to be inserted, flanked by DNA homologous to either size of the double strand break. This will then be used as a template, and the gene will be inserted into the gap.

An early issue with the CRISPR-Cas9 system was off-target effects due to the insufficient specificity of the 20nt guide sequence – other sections of the genome may have this sequence. However, this issue has been reduced via the utilisation of a ‘nickase’ mutant of Cas9. In these mutants, one of the two sequences required for cleavage is mutated such that it does not work, producing a single-stranded break instead. Now, to produce a double strand break, there is a 40nt of homology required.

Alternatively, by mutating both sites required for cleavage, cas9 can be directed to sit on a region of DNA. This could be utilised to tether a transcription factor to a gene that is usually switched off, for example.