Gene Editing - Cre/loxp (My Research, Vol. 2)

in #science7 years ago (edited)

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Pixabay.com


“Genetic modification”, “genetic engineering”, “transgenic animals” and “genetically modified organisms” are still very scary terms for many people. And while GMOs are not necessarily bad and those that are introduced to our environment are generally tested to be safe, there is still a lot of misinformation and fear.

Especially with the rise of CRISPR/Cas9 (about which I will make a post too), people are worried. What will happen? What devastating changes will researchers introduce to nature? Will we all die?

Well, yes, we will all die. But we’d do that without genetic engineering too, maybe even a lot faster.

Sketchguessolldie.png

Many breakthroughs show a promising future where we can fight cancer by simply reprogramming our own immune system, grow organs in animals (or even directly in a petri dish!), repair genetic diseases before the child is even born and more.

And I have a rather positive outlook on this, because of mainly one reason: Every researcher I met so far, online or in real life, professor or undergraduate student, aimed to do something to improve the world we live in. Sometimes for personal gain (I mean come on, how often have you put in hours upon hours of work for something completely altruistic?) but still with this as motivation.

What I actually want to talk about


Genetic manipulation is super old. The most basic one is probably selective breeding and has been done by our ancestors since the advent of agriculture. After that came a random approach, where scientists just dumped chemicals on plants or used radiation to create mutations and then checked what the effects were. @suesa

But even before CRISPR/Cas9, there were ways to manipulate the DNA of organisms - obviously, because CRISPR/Cas9 is only about 6 years old.

One of these tools for DNA editing is the Cre/Loxp system which comes from a bacteriophage. That’s a virus that only attacks bacteria.

Cre (“Cre recombinase”) is an enzyme that can direct the recombination of DNA sequences, which can be a deletion, insertion, translocation or inversion. For that, it needs so-called “Lox sites”, which are short regions on the DNA with a very specific look.

The system is a bit complicated to understand, so I’ll just show you how the deletion and the insertion works.

Deletion


For a deletion, the gene has to be located between two loxp sites. Upon activation, Cre causes cuts in the DNA which are then “sealed” again, which effectively cuts out the gene.

deletion.png

Insertion


Technically, insertion is just the opposite of the deletion, but the system favors deletion over insertion. To avoid a deletion of a previously inserted gene (which would be super annoying), researchers have created mutated loxp sites.

Each loxp site has a “core”, a “right arm” and a “left arm”. If one of these arms is mutated, Cre can still do its job. But if both are mutated, the “cut out” process doesn’t really work anymore, which makes the change stable.

Sketchinsertion.png

But why?


Sometimes, you just need to make a green fluorescent mouse. Other times, you want a mouse that can’t smell (fun fact: completely deleting a mouse’s sense of smell takes away all aggression and it’s a lot easier to keep several males together in groups).

Jokes aside, if you want to look into how a disease works and want to use an animal for testing, the animal needs to have that disease. Would be a bit pointless otherwise, no?

Especially genetic diseases can easily be induced by damaging or removing a gene. For others, you have to introduce new genetic material. Cre/loxp works so well, that it’s standard procedure in most labs that work with animals. And it doesn’t just work with mice! As far as I’ve seen, you can use it in every one of the usual model organisms (Drosophila melanogaster, zebrafish, Xenopus Laevis, C. elegans, …).

Sketchanimals.png

And the great thing is, it’s very specific. An animal’s genome (and our genome too!) usually doesn’t contain any loxp sites, which makes accidental changes (= “off-target hits”) very unlikely - in contrary to CRISPR/Cas9. And it’s less complicated to use too.

Some labs try a combination of the two: Using CRISPR/Cas9 to create a loxp site and then adding Cre to cause a recombination, specifically targeting the loxp site with Cas9, the possibilities are endless.

And you know what? It’s not the only way to change a genome. Even the Cre/loxp system has a lot of different ways to do things.


Sources:

Cre recombinase: the universal reagent for genome tailoring.

Accumulative gene integration into a pre-determined site using Cre/loxP.


Previously:

All about the Sperm-DNA - Vol. 1


Pictures are mine, if not indicated otherwise


stem-update-02-12-18.gif

GIF was created for me by @saywha and @atopy , rest of the signature by @overkillcoin

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Genetic engineering and modification is now beneficial for the world. Many foods are produced through this method.

How I love people who vote their own comments.... Easiest way to lose all my respect.

And the fact that diseases or deformations could be avoided is very pleasing!

Only recently did I learn that there is a difference between genetically modified and genetically engineered foods.

Well, yes, we will all die. But we’d do that without genetic engineering too, maybe even a lot faster.

Right XD
I'd love to see some lab pictures in a future post, I never had the time to research on how gene editing is actually done - I mean in practice.
This is such an interesting topic and i'm loving your series on it :-D

Gene editing is a bit anti-climactic and is mostly imagination and your own knowledge.

Example: Mix a drop of this colorless liquid with a drop of that colorless liquid. Put that colorless liquid into cells. Wait a week or so for the cells to divide so that you have more of them, then check if the cells got the desired genetic modifications. So it is important that you have the knowledge of what components are inside each of the previously mentioned colorless liquids and what they are doing, but otherwise it looks the same to the observer.

I know this because I do it almost every day in human pluripotent stem cells.

I'm wondering how mice without the sense of smell would react when confronted with the Maroilles, cheese from Northern France that smells even when ordered at the opposite side of the restaurant.

Special Like for Drosophila :D

This is educative, I've always been in interested in genetics, in fact if I had my way I would have studied genetics in the University. Thanks again

To bad you were born in Nigeria.

I recently saw a woman who was carrying her mechanical heart in a backpack. May be it's not so far when people will start using mechanical or bionic organ.

I think they help... GMOs I mean. They are the easiest way to feed the growing appetite of this universe. Especially war torn nations and the poor. Those who aren't allowed to farm or can't farm. How else will we feed them?

repair genetic diseases before the child is even born and more.
This is something, which is feared by so many people and I still can't understand why. The main argument is, that is is "unnatural" and would be the first step of getting rid of people with disabilities. This is just plainly wrong. Everytime you fight an illness with medication it is not really "natural" - fighting diseases even before the child is born is just the next logical step. Why would you not want to have a healthy child?

Maybe children with disabilities should be able to sue their parents for not preventing it out of sheer stupidity.

Very interesting how you explained this technique! The Cre/loxp system is a great method that has been used for many years and as you poninted out it is much more precise compared to newer modification methods such as CRISPR/Cas9. What do you think about its handling? I think in this regard CRISPR is advantageous. There is actually a modification of the CRISPR/Cas9 system called CRISPRi/a which does not edit the genes itself but modifies the gene regulation. I recently wrote about it, maybe you like to check it out.

I've just read three papers and a 125 pages ebook about crispr, I'm not sure if I can take it anymore 😅

@schay and @suesa send me any CRISPR/Cas9 questions you have. I use it every day to make new human stem cell lines.

Good to know! I'll be using it to create a loxp site in a specific gene of mice. I'll message you when it doesn't work :D

Wow! That is quite some research on CRISPR. I can imagine, you had enough.

Genetic engineering. Is that the thing that if I'm an engineer, my son has a 50% chance to become an engineer - and if I get a baby with another engineer, our child will become an engineer with 100% probability?

Exactly.

Yes, yes but, could you make a tomaco?
Nice drawing :)

Tomaccos are actually super easy, highly toxic and have been crated before!

Wow this is very interesting to know that genetic engineering promises us with an amazing future,with this we would be able to conquer cancer. I am grateful to God for this knowledge of genetic engineering..

This is highly controversial as the genetic changes could affect sperm or egg cells, meaning the genetic edits and any bad side effects could be passed on to future generations.
On the flip side , this is a very informative post which illustrates the potentials of gene editing to treat or prevent human diseases.

Really nice write up on genetics it is really educational.

sir.
your post really good.i like your post,and thanks yor shearing.

Additionally, I'm not a man.

I am just here to laugh :D

Thanks for the knowledge update. I will anticipate your CRISPR/Cas9 post. Thanks again

I read it all, honestly, barley understood anything.
I'll sit and read it again tomorrow at the office, I guess it's because English isn't my native language so It's a bit harder for me. I'll read it with checking words tomorrow, just don't have time. :)

Genetic mutation as an area of research is highly academic. I salute your courage for delving into it.. Thanks @suesa

Quite insightful. Thanks for sharing this.

The quest to find ways of making organ replacement and surgery easier is really pushing more researches to surface.

I just read about the implanting of human cells in sheep some hours ago. This post is another one elaborate on genetic research.

I would say it is a bit complex though. Hoping to read more on it.

Thanks. It's a worthwhile read.

Gene editing has been drawing some attention lately. Maybe, very soon, the CRISPR gene editing tool would get so advanced that scientists would use it to tweak genes and make us live longer. Or even reverse the process of senescence.

Nice piece ma'am

Hi @suesa modification of genetic disease are very important now, because there are many ways to avoid it . I must say this is great what you have written.

Jokes aside, if you want to look into how a disease works and want to use an animal for testing, the animal needs to have that disease. Would be a bit pointless otherwise, no?

And i believe there's a way to test that the animal actually suffers from such disease.

And can an animal has same disease as humans?

The scince reserch on it. i like it. thanks for the share.

Am I right in saying that a company in the States has been granted Human trials for testing genetic manipulation?

I had read in The Near Future Report that this was possible and would be a very valuable investment for anyone looking to get into it. Obviously, investments aren't the aim of the game, the benefits to humanity are massive when it comes to curing genetic diseases.

What company? Where in the states? Testing what kind of manipulation? Your question is a bit vague.

I just asked if you knew of the company. It is called Illumina after finding the email.

Cool article, but the Cre/Loxp system will (most likely) never be used in a clinical setting for humans, at least not in the United States or anywhere with a decent regulatory body. Perhaps the biggest reason is that extra, non-native, sequences of DNA are being inserted into the genome (loxp sites). Even after removal with Cre, there is a "scar" that leaves behind one loxp site in the genome, which is considered a no-no.

As for wild-type CRISPR/Cas9 for the clinical setting, I think it inherently is dangerous to use because it can have a lot of unintended, off-target effects. My opinion is that the current state of CRISPR/Cas9 is good for quickly and cost-effectively investigating genetic modifications, but a more specific version or a new method of gene editing that is more specific would need to be used for high success rates, and low safety concerns, in a clinical setting.

yes it is the time

You're right.

the science....
reserch on it..
love it

No way you read this in under 2 minutes. Spammer.

resteemit done...

Maybe read before you resteem, because I doubt you read this in under a minute. Aaaand that makes this comment spam.

I am really sorry dear...
please forgive me...

steady the more successful, greeting friends steemian