How exactly does CRISPR-CAS9 insert new genes?

Submitted by AutomaticAd1918 t3_z67gnl in askscience

I understand the very basics of how CAS9 enzyme cuts off a gene with a guide RNA to help it locate,, but what's still unclear to me is what we do with the DNA breaks

• If we let the cells repair it by themselves, will they not just remake the segment we just cut off? • If we insert a new gene, how exactly do we deliver it? Does it come with the CAS9 and guide RNA complex? Or do we use another enzyme to deliver it separately?

I've just started learning about this topic so I'm sorry if my question seems very basic. I've tried searching online it but so far I've only gotten things like, "We insert the gene..." "We deliver the new gene..." or "The new DNA segment is delivered" without specifying how it was delivered

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Smeghead333 t1_ixzx8i5 wrote

When you insert the enzyme and guide RNA, you also add a bunch of copies of short DNA sequences that match the region being cut, but that include the change you want to make. Then when the cell repairs the break, it's likely to grab one of these synthetic sequences to serve as the repair template, and boom. Change made.

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Aescwicca t1_iy0l6hd wrote

Ok... but how do you make the synthetic chains?? Like really REALLY small tweezers?

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YouDrink t1_iy0ppsc wrote

You can make them via chemical synthesis.

There are four nucleotides that make up DNA (A, T, C, G). If you want to make a sequence such as ATGCCGA, you start with A, react it with a bunch of T and wash, react it with a bunch of G and wash, etc to make your sequence.

To avoid uncontrolled polymerization, you use nucleotides that are capped, so they can only react once. This requires an intermediate step to activate them for reaction. So you start with A, decap it, add a capped T, wash. Then you decap, add your capped G, wash, decap. This let's you control exactly what your final sequence looks like.

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Seicair t1_iy0qi83 wrote

What length sequences can be practically made with this method?

Edit- I’ve taken college senior level biochem classes, for background.

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YouDrink t1_iy0t0ll wrote

Very common for 20-60 nucleotides, but can be done for 3000 nucleotides.

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CompMolNeuro t1_iy14ulx wrote

Just extra fun information. There are other gene transmission methods that can carry up 30 or 40 thousand nucleotides, like repacking a retrovirus, though it's not organism wide and the research has probably been set aside since I last saw the inside of a lab.

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MarsLumograph t1_iy1jbkt wrote

But they are not talking about gene transmission? They are talking about DNA synthesis. How many nucleotides can you add with that method.

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Iniquitous33 t1_iy1yhto wrote

That method generally stops being efficient as you get to the higher double digits, but you can stitch those double digit length pieces together after they've been synthesized using a different chemical process. That makes the manufacture of these 40Kmers or really any conceivable length possible - though certainly not practical or economical.

Manufactured oligonucleotide material is very expensive relative to small molecule or even antibody medicines. The industry is working to solve that, as it's a relatively newer type of medicine, but whoever can figure that out will dramatically open up treatments for rare disease and personalized medicine, as well as bringing the cost down for tons of quality of life treatments that exist and are great, but are cost prohibitive vs standard treatments that work but only ok, or have side effects that suck but aren't bad enough to justify a treatment that's 50x more expensive.

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MarsLumograph t1_iy2ktcy wrote

I don't think DNA synthesis is the main limiting factor for gene therapy, but delivery and safety.

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Iniquitous33 t1_iy38r0x wrote

I was actually referring to ASO, siRNA, and other direct oligonucleotide therapeutics. These are viable right now, safe and effective. Gene therapy is kind of "next in line" as it were, but as you implied still has some major kinks to be worked out. Though I believe it will be relatively figured out given time. I'm pretty excited to see what the next decade holds for the field at large.

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Outrageous_Cry_5945 t1_iy2akdm wrote

Also AAV, adeno-associated viruses . . . (but not preferable in a clinical setting/application usually, since they apparently can sometimes elicit immune responses and may contribute to the deaths of some patients historically in gene therapy trials https://www.nature.com/articles/s41587-020-0642-9 High-dose AAV gene therapy deaths, liver dysfunction, sepsis . . . )

Edit: I suspect that if we can use nanolipid particles as vectors, that may be more safe instead of AAV vectors.

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Jman9420 t1_iy0ulaw wrote

The synthesis of these chains (referred to as oligonucleotides) is usually done by companies that specialize in the process. Most of the time the sequences they synthesize are less than 200 base pairs in length. However, there are a lot of ways to ligate multiple of these fragments together and so you can purchase longer sequences that are a few thousand base pairs in length from these companies.

Depending on the genetic change that is being attempted it can vary what length of synthetic DNA is needed. Often a scientist can use oligonucleotides that are only 20-60 base pairs long along with polymerase chain reactions (PCR) to synthesize larger fragments with the needed modifications. Other times it is necessary to have an entire gene or sequence of genes completely synthesized to be compatible with the host of interest.

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corknut1 t1_iy1q8re wrote

The practical limit is a multiplicative function of the error rate of the chemistry involved.

If the %succes of adding a new base to the growing chain is X, and the length of the chain is N, the overall % of success for a given length is X raised to the power of N.

So for example;

If you have a 98% success rate of adding a new base to the growing chain (ie 98% of the millions of chains you are extending successfully extend),

by the time you are adding base 100, you have 98%^100 or ~13% of the original starting material as 100 base long oligonucleotides.

The shorter chains are discarded during a purification step after you've finish the addition of bases.

Practical limit of synthesis length is usually under 100 for this reason, but it is then possible (as someone mentioned in another comment) to join these together in a separate chemical process (ligation, not to be confused with the surgical definition)

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theartificialkid t1_iy1vnzi wrote

Can you use PCR to reset? (ie take your small yield of correct sequences and multiply them so you can start again at the top of the yield drop-off curve with part of your sequence already in place)

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InaMellophoneMood t1_iy1ymsr wrote

Why would you do that when you can just stitch together many chunks using modern assembly techniques? That way you can use synthesis in the cheap, high yield part of its curve, and plasmid replication/purification to yield large quantities of your sequence for even cheaper. Fussing with super long synthesis with the flaws of existing chemistries doesn't make sense when it's more time and labor intensive than assembling them from medium length synthesis.

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corknut1 t1_iy4z7or wrote

Not really - the initial base needs to be attached to a substrate (e.g. CPG) or support. It's only removed from this substrate at the final step once you have finished extending the chain.

Think of the support as the thing that keeps your DNA in the bottle when you're doing the chemistry; during the synthesis you're repeatedly adding chemicals then washing them away.

If your DNA product isn't firmly attached to something during this process, it's going to get washed away too.

It's conceivable you could remove the DNA from the support, capture it, amplify it with PCR, then reattach to support to continue the extension, but the re-attach part would be very difficult - you'd be dealing with a long floppy chain and trying to attach one end to a solid anchor via some unknown complex chemistry. There would be side-products, loops, breaks, etc. to deal with. Someone has probably tried it, but it's not something I've ever encountered.

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mkovic t1_iy0uq7y wrote

My experience was with peptides but the steps are similar. The chains we were making were 15-20 amino acids and, in a university lab setting, it took a few days to make a batch. Making the chain longer would extend that time pretty proportionally. The yield also goes down with each added step.

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heresacorrection t1_iy1gkuv wrote

Practically they don’t do more than a few thousand (also different platforms probably have different physical limits). But it would probably make more sense practically, if for example you were building a synthetic genome, to do it in chunks. Then after ligate the chunks together afterwards.

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toolemeister t1_iy1bkhy wrote

How is this physically done, as in equipment/machinery etc.? I understand the abstract concepts but I'm more interested in the engineering!

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corknut1 t1_iy1rzwq wrote

It's essentially done with robotic pipettes, though the ones used in DNA synthesis are very specialized.

It can be done with hand pipettes, but it would be a slow and error prone process.

The materials are not exotic and can be purchased from chemical supply.

Typically the starting material is a (population of) a single DNA (or RNA) base bonded to a glass bead (cpg), and the chemistry is done by washing the beads with a series of chemicals, following a repeated recipe. The recipe is identical for each base added to the chain (excepting the base itself of course).

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RoundScientist t1_iy7qqoi wrote

This is what was described to me in a lecture.
You have beads of resin which you can chemically link chemical derivatives of nucleotides to.

  1. You link your first nucleotide to the bead.
  2. You flush out excess nucleotides.
  3. You add a reactant that makes all unoccupied parts of the resin non-reactive for the kind of chemistry you're doing.
  4. You remove the protective group that prevented the nucleotide derivatives from linking up with one another.
  5. You have unprotected nucleotide derivatives chemically linked to your resin beads. You now start back at step 1 - only this time, the new nucleotides you add bind to those from the previous cycle. After all, these are the only positions left where you can have linking reactions.

And then you just loop the process and by picking the order of reagents, you determine the nucleotide sequence. The inactivation step prevents the further growth of oligonucleotides which "missed" a step.
Once you're done, you perform a reaction which splits the oligonucleotides off the beads and purify them by length. I think usually via chromatography.

Which means that you have a machine with several input liquids and valves (solutions of your reagents, solvent, one reservoir & valve for solutions of each derivative nucleotide), a reaction chamber with beads and a valve for removing liquid/trash collection.
Then it's just a matter of sucking in/out the right liquids in the right order, with long enough pauses for the reactions to take place. Possibly with heating.

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nmezib t1_iy2fccm wrote

And in case anyone is wondering, research groups themselves generally don't do this. They get a big company like IDT or Sigma to do it for them for really cheap.

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New_Concert_4315 t1_iy14l2p wrote

Doesn't the 5 prime of initial A that gets decapped bind to the 3 prime of the T? If not, why?

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corknut1 t1_iy1setu wrote

The initial end is bonded to a "support" (glass beads called cpg are typical) and only cleaved from the support once the entire chain is grown. The cleaving process also caps this end.

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Germanofthebored t1_iy1cfcb wrote

Good question - one reason is that the chain isn't flexible enough to make such a tight turn. Still, a free nucleotide building block could hydrogen bond to a growing chain. But that would only be 2 or 3 H bonds, and thus much too weak to stabilize the complex

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heresacorrection t1_iy1ez3m wrote

The “cap” only allows 3’ additions OP probably simplified the wording it’s not an actual 5’ cap (as you hear about in mRNA), it’s a chemical blocking done by modifying the 3’ end of the chain. The 5’ is never exposed.

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spastical-mackerel t1_iy1fhr2 wrote

You head down to EnzymeVille, the squalid yet vibrant neighborhood in your dystopian mega-city where criminals, fugitives, and other desperados eat noodles in the rain and furtively sell body parts and secretions and buy bespoke DNA for purposes as varied as the human condition but which are never spoken aloud.

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wanson t1_iy2uc5j wrote

It’s just an oligo. We use them as primers all the time in PCR. You just decide what bases you want and order it from a company. Small 20 base pair primers cost about $3.

They’re made by a chemical process and shipped dried (lyophilized). We just dissolve them in water or buffer.

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HotDadBod1255 t1_iy36fb7 wrote

Oligos used for CRISPR-Cas9 are extremely expensive. They're usually larger (60-120mer) and have to be much more pure to avoid off target editing.

They're currently one of the biggest bottlenecks in the gene therapy field since we can't make enough of it fast enough and with high enough purity.

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wanson t1_iy3hkaj wrote

They can be. It depends on what you're doing. For me, I am just correcting SNPs in cell lines so 50-60 bp is enough and they're only a couple hundred dollars or so.

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HotDadBod1255 t1_iy3l7ih wrote

Right, totally different needs here. I use them for in-vivo gene editing, so purity and precision are paramount.

Like most things out there, when you need super high purity and quality, it's gonna cost you a lot. In this case oligos are already a pain to make so it's ridiculously expensive.

To give you an idea, around 2000mg of the oligo I use as guide RNA will cost in the $2M range.

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22marks t1_iy1z5m4 wrote

Are there just random sequences always floating around for repairs? Forgive the analogy from a layperson, but is there any form of checksum? How does it know it needs to grab that sequence, especially since it’s not even the original sequence it’s replacing? Simply because the ends match like perfect “puzzle pieces” and it’s like “good enough?”

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Smeghead333 t1_iy20oib wrote

Normally, when a break happens, there's another copy of the DNA sequence in the cell - remember you have two copies of each chromosome: one from your mom and one from your dad. So the repair mechanism looks for another similar sequence and copies it (oversimplifying here) to patch the hole.

With CRISPR, if you inject a few thousand or million copies of the altered sequence you want, the odds are very good that the repair system will grab one of those instead of the non-altered sequence on the other chromosome.

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CrateDane t1_iy4yv1k wrote

>Normally, when a break happens, there's another copy of the DNA sequence in the cell - remember you have two copies of each chromosome: one from your mom and one from your dad. So the repair mechanism looks for another similar sequence and copies it (oversimplifying here) to patch the hole.

HDR is mainly active in S and G2 phase, where you get up to four copies of each chromosome - two maternal, two paternal. That provides additional templates for repair (or let's say a stalled replication fork ripped both paternal sister chromatids apart - you then still have two maternal templates available).

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Hour_Situation_9469 t1_iy2ujzu wrote

Are you saying they insert engineered Copy number variants with the alteration contained??

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FogeltheVogel t1_iy01ut5 wrote

When DNA experiences a double stranded break (the type that CAS9 makes), there are 2 methods a cell has to repair it.

The first is the sloppy one, called non-Nomologous End Joining. The machinery for DNA repair can't really do anything with blunt breaks (the type that CAS9 makes), it needs ends that stick out a bit. Called literally Sticky Ends (if they overlap). Sticky vs blunt ends. So the first step is enzymes that remove some nucleotides from each end to make them sticky. After that, other enzymes come in that take these sticky ends and extend them into each other, repairing the break. The problem here is that some nucleotides get lost, and some random ones are added. This usually breaks the gene. When using CAS9 to knock out genes, this is sufficient.

The second method is what's used here. It's called Homologous Recombination, and it is not always possible to use. In essence, it uses the other chromosome as a template to accurately repair the DNA. Under normal conditions, this can't be used because the other chromosome is not readily available to serve as a template. During CRISPR treatments to insert a new geme, we provide a piece of DNA along with the CAS9 and it's guide RNA. This piece of DNA is the "new" gene, but it isn't incorporated like you originally thought. Instead, it is specifically designed so that the 2 ends of this piece of DNA are perfect matches to the 2 sides of the break made by CAS9. The DNA repair complexes will see thus use this as a template to 'repair' the break perfectly. Only in this case, they include extra nucleotides that were never part of the original strand.

I hope this helps.

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CrateDane t1_iy060yz wrote

NHEJ can also be used for insertion, in strategies such as homology-independent targeted insertion (HITI). Because the ends being joined don't have to be homologous, you can co-deliver a linear DNA fragment and have the end joining pathway insert that where the double-stranded break was made.

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FogeltheVogel t1_iy06eoh wrote

Is there any way to guide this process? It feels like this would have a rather low chance of actually happening.

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Cleistheknees t1_iy0u6dt wrote

Preface: the last “i” in HITI is actually for “integration”, not “insertion” as stated above, in case you wanted to google around for more info.

You guide the process in the same way. The donor sequence used (theoretically used, anyways) in HITI is capped by the same sequences that gRNA-Cas9 is pointed at, and in fact is exceptionally accurate. The idea behind HITI is that it isn’t limited to actively differentiating cells.

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deisle t1_iy24kmd wrote

You're right, all of these processes require that you have all the bits in the right place at the right time and the cell does the right thing and no enzyme messes up too hard. So you shove as much stuff in as you can to maximize your chances When I would try to insert a mutation in a zebrafish, I would inject hundreds of fertilized eggs at the single cell stage, let them grow up, and then take a tail clipping to genotype. I'd be lucky if I got a couple successful mutations from those hundreds of eggs. It's definitely a numbers game.

Caveat: this was like 6 years ago, when it was relatively new. Success rates have likely gone up as the technique has been refined but general principle remains

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worotan t1_iy0hgwu wrote

That’s so clear for a complete laymen who is fascinated, thank you. It makes perfect sense.

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Plantpong t1_iy0b0x7 wrote

Agreed with the above! NHEJ is often used for the insertion of 'random' nucleotides for generating gene knockouts. HR can more efficiently be used for targeted insertions when also introducing a repair template along with the CRISPR complex that has homology arms that match the target splice site.

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gthing t1_iy1ljxx wrote

If my 23 and me shows me I have certain genes that are associated with higher risk for X,Y,Z - are those theoretically then curable with CAS9? Are the genes even understood enough to say if we switch one off it's not going to have some cascading weird effect or even that it will actually cure you? Last question: how long in your wild estimate until most everyday gene disorders are routinely cured during childhood?

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wilnyb t1_iy27zw0 wrote

A major issue with correcting genes in adults is the the large number of cells that needs to be corrected. Let's say you have a muscle disease, hitting every single muscle cell in you body to correct an genetic disorder is very complicated. As of right now, this is easier with blood cells. You can isolate hematopoietic stem cells, that you can edit and then reintroduce in a patient. Those cell will repopulate the immune system.

Every disease is different. For many diseases it might be enough to correct 10% of the cells for a patient to be able to live with the disorder. Some of those examples already exist today. Some more complicated genetic disorders we might never be able to correct (at least in our life time).

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HotDadBod1255 t1_iy36nd2 wrote

Unfortunately there really isn't much clinical data for in vivo gene editing, the only results so far are from two clinical trials from Intellia Therapeutics. Their results are really promising, but so far they've only shown they can do gene knockout in liver cells, which is pretty limited in scope. Hopefully them and some others are working on other ways to perform gene editing.

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-Metacelsus- t1_iy0cbl2 wrote

I do this regularly as part of my research. Here's how it works:

I make a bacterial plasmid that contains the DNA that I want to insert. On either side of this DNA, I have an additional 1000 bases of DNA that has the same sequence as my target site. These are known as homology regions. I can assemble this plasmid using a method such as Gibson assembly.

I then introduce this plasmid into the cells, along with another plasmid expressing Cas9 and guide RNA, using electroporation. The Cas9 and guide RNA cut the target site. The cell then tries to repair it.

The usual repair pathway is called non-homologous end joining, which simply sticks the DNA back together. This is not what I want. However, cells can also repair DNA through homology-directed repair (HDR), where they basically look for similar sequences and swap them into the cut site.

When cells perform HDR, they can use my plasmid to perform the repair because it has the homology regions. Once this happens, the DNA sequence becomes inserted into the target site.

For a good intro-level review of this, I recommend: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901406/

(Note that there are other ways being developed to do edits with CRISPR, I'm just explaining HDR)

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pitchapatent t1_iy0kkcz wrote

Wonderful reply! I would just emphasize/clarify that the HDR template (the DNA that you want to paste) doesn't actually get incorporated into the cell's genome. It merely serves as a guide, allowing the cell's repair machinery to copy the sequence info into the genome. I know you know this, but it's one of the most counterintuitive and easy-to-miss elements of the pathway.

For another entry-level resource on CRISPR everything, I would recommend CRISPRpedia - I've linked the "technology" page and it has an HDR section. But it also glosses over the it's-not-actually-pasted-in aspect that I addressed above. Even worse, this video shows something at 3:00 that simply does not happen (repair template being physically pasted into the genome).

This video does show an accurate depiction of the repair mechanism, with the good stuff starting at 1:00. Although this is not a CRISPR-specific video, the mechanism is very similar. Just think of the pink DNA as the repair template - the scientist-provided sequence-to-be-pasted. This is the single best resource I'm aware of that succinctly addresses the question posed by /u/AutomaticAd1918

OP, since you asked about how these things get into cells, you should check out the "Delivering CRISPR therapies" section of this CRISPRpedia page on genetic medicines. In brief, scientists can use viral vectors (widely used in gene therapy), lipid nanoparticles (same tech as COVID vaccines), or physical/mechanical means to get large molecules into cells. Although that page focuses on getting the CRISPR enzyme into a cell, the same approaches work for delivery of the HDR repair template (the DNA to be pasted). Delivery is a major challenge because cell membranes are fiercely dedicated to acting as a barrier, and they're very effective at preventing transit of larger molecules. Traditional small molecule drugs don't face the same delivery challenges because they can "slip through the cracks" and enter cells via diffusion.

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trijammer t1_iy2l7md wrote

Thanks for this clarification. The video of the repair process is great.

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HappyAntonym t1_iy0ruo3 wrote

Woah, great explanation! It sounds like you're basically creating a Trojan Horse out of DNA.

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TheDurrrmanNeighbor t1_iy11k58 wrote

I remember back in the day there was an episode of a cartoon where they tried to pull off a bank heist. The character spent time devising a plot and the simplest plan without fail was to create path.

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[deleted] t1_iy2gm7i wrote

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-Metacelsus- t1_iy34e5r wrote

> What I don’t understand is how this would work for an entire body?

Your understanding is correct, because it doesn't work for an entire body, the efficiency per cell is not nearly good enough. If you want a full-body edit you would have to edit stem cells, select the edited cells you want, and then use various embryology techniques to put the stem cells in an embryo and have them develop into a new organism.

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[deleted] t1_iy34o2b wrote

[deleted]

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CrateDane t1_iy50gdd wrote

Depends on the gene defect; if it's enough to edit 3% of your liver cells to produce an important protein to circulate in your blood, then it's pretty reasonable to expect to cure that disease with (non-germline) CRISPR/Cas.

If it's something that needs to be fixed in 100% of a certain cell type, especially non-proliferating cells, then that's going to be very tough. And if it's something that acts during development, then fixing the DNA in an adult would do nothing (the body has already been "built" with the wrong "blueprint").

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Astavri t1_iy16nnp wrote

You transfect the cas9 plasmid and not deliver the enzyme itself?

Do people not typically send the enzyme itself using electroporation? I was unaware of this part.

Basically all you are doing is a transduction/transfection then, with all the necessary genes being sent through.

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-Metacelsus- t1_iy19ucd wrote

Doing it with the cas9 plasmid is cheaper and works nearly as well. There is a greater chance of off-target edits though. But yes, if I was doing it clinically I would use the ribonucleoprotein.

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Astavri t1_iy1ba6r wrote

That's clever. I never thought about that as an option.

Do you have any publications or sources for doing it this way? If it's not any trouble.

I don't use crispr for anything at the moment, but there might be something I want to try it on. This 100% seems like the better option. And you could reuse the same plasmid with the cas9 and guide correct me if im wrong, and change the genes on the second plasmid if you want to try introducing a different gene in the same region?

Is it Ecoli or mammalian cells you are editing?

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CrateDane t1_iy4zwth wrote

You have multiple options. You can provide the Cas9 gene in DNA form, but also as mRNA. Or simply the protein, usually pre-assembled with guide RNA into a RNP.

Even the Cas9 DNA delivery has multiple options - viral vector, plasmid transfection, it all depends on the use case.

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inblue01 t1_iy0cn7w wrote

Some good answers here, but one piece is missing : screening!

You are correct: in most cases, the cells will repair the genome in a way that does not suit your downstream applications. It's a matter of probability : one in many cells will, by chance, repair your DNA with your template, or do the desired mutation.

One step that you need to do is to isolate clones (single cells), let them grow to a decent population and then screening (by sequencing and functional testing) these clones to determine which one has the desired insertion/mutation.

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fertthrowaway t1_iy1s8at wrote

With repair template and active enough homologous recombination, you can get like 99-100% correct edit efficiency in many organisms.

If you rely on NHEJ after the nuclease cut, then you basically have to screen through repaired DNA craters though, yeah.

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WiwaxiaS t1_iy202zz wrote

Ah yes, that's why indicator genes may be co-introduced, like GFP for instance, and sure functional testing for the gene products themselves can also be done.

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Ph0ton t1_iy0nzod wrote

To be clear, CRISPR-Cas9 does not insert new genes. It's a nucleoprotein complex which simply creates a double-stranded break that allows for the opportunity for DNA to be inserted during the repair pathways. For a gene to be inserted, it must be first localized to the break site, then the right pathway must be initiated to insert the gene, and finally the whole sequence must be inserted to be functional.

In therapies where the patient's own cells are removed, edited, grown, and then transplanted back into the patient, the unlikelihood of a gene to be inserted correctly doesn't matter as much. With millions of cells, we only need a percentage to take up the gene, and then we only need to screen for those lucky few to culture for the transplantation. This is also complimented with cell culture techniques where we can arrest or cycle the cells in specific modes where it favors the better repair pathway. Delivery is also easier as we can use electroporation to insert (relatively) large payloads of genes or machinery. The same applies for edits within other cells; if we can culture them then it is trivial to "insert" a gene.

This is drastically more complex for editing in vivo, where we want a pre-existing population of cells to take up those inserts. For this challenge, we need other tools, such as CRISPR-Prime, PASTE, or different "flavors" of CRISPR-Cas proteins; from nature or designed ourselves. This is still a work in progress, and even delivering a gene of significant size is a challenge.

Generally speaking, the mechanics of the insert are extremely specific to the domain and objective of the research or therapy. If you are wondering about a specific development I'm happy to look at the paper to parse it for you.

Source: Former CRISPR-Cas3 researcher

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WiwaxiaS t1_iy20haa wrote

Oh yeah, I was actually wondering about that. Compared to where one can just keep cloning the successful survivors as in in-vitro, in-vivo would require far more accuracy. Didn't know those new developments were also underway. Very cool.

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[deleted] t1_iy1gatw wrote

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Ph0ton t1_iy1iimh wrote

Given that the fusion protein is usually characterized by chromosomal translocation, such abnormalities represent a huge restructuring of genomic material, and would not be a good target for a precision tool like CRISPR-Cas9. You could theoretically use something like CRISPR-Cas3 to shred the extraneous material, but to what end? I would think that such a cell is not worth repairing and should instead be targeted for destruction through other therapies.

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AutomaticAd1918 OP t1_iy2d7dq wrote

Thank you for your reply! May I ask what are the Pros and cons of using PASTE? I heard it's relatively new but it's apparently better than CRISPR in some ways

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Ph0ton t1_iy2ptbh wrote

It's a new technique that iterates upon existing work with integrases and fusion proteins in concert with CRISPR-Cas9 nickase to deliver huge packages of DNA. This existing work is still relatively new but is extremely promising, so PASTE has realized some of that potential. The pros are obvious: the ability to deliver large sections of DNA into multiple loci, dodging some of the deleterious effects of cellular repair pathways. As for cons, like many newer techniques, it requires expertise and development of various facets of the the insertion machinery. The promise of cas9 is any lab has the resources to develop a short guide RNA to make an edit, and they have a wealth of mature techniques to utilize said edit in most kinds of cells; also it's so easy a high schooler could run an interference assay (and they do).

As with any emerging tech, there will be unrealized challenges as it is deployed in various organisms, through numerous transfection techniques, but I wouldn't deign to speculate on those cons without a thorough review of the biochemistry (and other labs putting it into practice).

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keenly_disinterested t1_iy0cavu wrote

I strongly recommend you read The Code Breaker, by Walter Isaacson. It's about Jennifer Doudna's work in the field, and clearly describes the research and processes involved in the most layman of terms.

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ocular__patdown t1_iy0408t wrote

> >• If we let the cells repair it by themselves, will they not just remake the segment we just cut off?

They will just try to glue the ends back together if you dont add a template for it to insert.

>• If we insert a new gene, how exactly do we deliver it? Does it come with the CAS9 and guide RNA complex? Or do we use another enzyme to deliver it separately?

Usually you supply the template at the same time you supply the Cas9. This can just be DNA. That way when the DNA is cut the template is already available for insertion.

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CrateDane t1_iy05nar wrote

>• If we let the cells repair it by themselves, will they not just remake the segment we just cut off?

Depending on the strategy used, they often do just that. Afterwards, you can just select the cells that used the repair template with your insert.

>• If we insert a new gene, how exactly do we deliver it? Does it come with the CAS9 and guide RNA complex? Or do we use another enzyme to deliver it separately?

Traditionally it's a separate piece of DNA we deliver. But prime editing actually delivers the template as part of the guide RNA. That only allows fairly small edits though.

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WiwaxiaS t1_iy1w4fy wrote

It doesn't. The CRISPR-Cas9 complex itself only cleaves and removes a designated portion of DNA, and another matching/desired short linear DNA sequence is introduced into the vacancy left by the double strand break (DSB) induced by Cas9 cleavage and connected through ligase activity, either through homology-directed repair (HDR) or non-homologous end joining (NHEJ). The latter process may induce new insertions and deletions (indels) around the joining sequence due to sticky ends being required to form and thus cause inaccuracies, so if it can be done HDR would be preferred for introducing new genes. Typically though the processes would all be separate, the tools would all be packed in one synthesized plasmid. An internal ribosome entry site (IRES) sequence may be included in the plasmid for translation and expression of distinct gene segments after transcription, if more than one gene is to be introduced, or even just during the initial phase where different components may need to be translated at the same time. In in-vitro or in vivo studies sometimes an indicator gene like the green fluorescent protein (GFP) gene can be co-introduced to check whether the edit was successful. Overall as you may guess it can be trickier to introduce new gene elements compared to just cutting and inactivating genes.

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HotDadBod1255 t1_iy35sti wrote

I think most answers here are covering the ex vivo CRISPR-Cas9 applications. Just gonna throw in a thought on in vivo since that's what I work on.

As others stated, CRISPR-Cas9 only cleaves and removes DNA, it does not insert new DNA. It does this by using a strand of guide RNA which has a nucleoside sequence matching that of the target DNA site (there's a lot more to that subject as well). This has been done successfully in vivo by Intwllia Therapeutics, there are a few other companies that are beginning to does patients but don't have results yet.

I'm order to insert new DNA, some other tool is needed. But to date, we have not done this in humans! The most promising technology to do this right now is an AAV delivery mechanism.

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AutomaticAd1918 OP t1_iy3cc1a wrote

Ooh I see based on the answers, I've also understood that instead of "inserting" genes, in general we really just break off a part of DNA, then allow the cell to repair it and just hope it repairs it in a way that we would like or we can give a template so the cell can more likely repair it in a desired way?

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HotDadBod1255 t1_iy3g7wt wrote

Correct! In ex vivo applications we really need an insertion mechanism.

For in vivo, there are a lot of diseases we can treat by just knocking out the gene and not inserting anything.

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AutomaticAd1918 OP t1_iy3gt52 wrote

Ooohh I see how are genes knocked out or shut off? Do we introduce another protein to stop the cell from repairing it?

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HotDadBod1255 t1_iy3lmrj wrote

Nope, it's just the Cas9 protein and the guide RNA. The guide tells Cas9 precisely where to make cuts in the DNA. The part that's cut is naturally removed and digested by the cell. Then when that happens, the cell's natural repair mechanism takes the two open ends of DNA and connects them together.

As you might guess there's some work required to figure out dosing. To low of a dose and you probably won't edit enough cells, so the un-cut gene will persist. Too high of a dose and you're probably going to harm the patient or cell.

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bioprog t1_iy01b2i wrote

There's two general pathways for repair of a double stranded break. You should look into both and this will answer your question. The first is non homologous end joining (generally this leads to the introduction of indels), and the second is homology directed repair (which is the mechanism used to insert new segments of DNA near a double stranded break).

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LastVisitorFromEarth t1_iy0cb9p wrote

You have to understand that there isn’t much intent behind these things. It’s just.. probability.. Chemistry. These are just gradients. But they are in such high numbers that stuff will happen. And you can manipulate how likely it is that stuff will happen.

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TheSeaOfTime t1_iy0ksax wrote

It doesn’t. It removes the target gene and cuts it into small segments. If there is no replacement, the cell will repair the DNA, most likely getting the order wrong and ending up with useless code. If there is a replacement gene, the cell will insert the replacement at the break and maybe insert a few of the junk gene pieces.

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Ambitious_Power_1764 t1_iy365ms wrote

It doesn't matter how it works. It will never be allowed by our corporate / government overlords to defeat the three most critical issues facing humanity.

  1. Aging
  2. Disease
  3. Death

If our corporate / government overlords actually cared about progressing humanity. Every single penny and resource of the entire governments of the entire world would be poured into solving, curing, and eliminating the big three. Aging, disease, and death.

There is not a single human being on this planet that is not threatened and will not ultimately face these three existential issues that have been ongoing and uncured since the dawn of human existence.

CRISPR-CAS9 can solve those three problems. However, our world corporations / governments are too short sighted to see the benefits of allowing such progression. They will silence, discredit, and eliminate any progression towards curing the big three.

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