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Seicair t1_iy0qi83 wrote

Reply to comment by YouDrink in How exactly does CRISPR-CAS9 insert new genes? by AutomaticAd1918

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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