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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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