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OfflineKrombopulosM
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Point mutation
    #24870839 - 12/27/17 06:56 AM (3 years, 4 months ago)

Does anyone have any good info on teks for point mutation? Specifically methods for removing or adding nucleotide bases. I feel like I'm gonna have to hunt down sequencing equipment pretty soon. They are under 3 grand now which is pretty affordable imo. Thanks for any help in this area.


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Offlinelmao
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Re: Point mutation [Re: KrombopulosM]
    #24871065 - 12/27/17 11:05 AM (3 years, 4 months ago)

If a sequencer would cost less than 3 grand then I'm sure every university lab would have multiple instead of one sequencer for the whole faculty as it is right now.
https://www.ebay.com/itm/Illumina-HiSeq-2000-neXtGen-Genome-Sequencer-with-Computer/182640191050?epid=1452359973&hash=item2a8634264a:g:AOgAAOSw2iNZYFZl

A bit more than 3 grand if you ask me.

It's hard to really comment on your post. I'm sure there isn't a "tek" on how to do point mutation. But I'm sure there are a few dissertations, because people spent 3 years with 40+ hours per week to find a single working point mutation and backbreed it to some elite cultivars.

It is really nice that you are interested in this kind of stuff but you need a whole molecular lab set up. PCR, gel electrophoresis, laminar flow, the right DNA primers....it all involves so so much trial and error that it isn't yet possible to do at home.
Give it another 10 years and we can all do our own Crispr/CAS9 at home.

For now, if you really want to improve certain attributes, go out there in the forest! Try to find interesting individuals, preserve them, grow them, further select them.

Maybe start slow, put 2 indidivual spores on a petri dish, check the outcome. But first of all you need kinda all the basics about genetics, rules of inheritance, alleles, dominant, ploidy, sexual reproduction of basidiomycota....


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Re: Point mutation [Re: KrombopulosM]
    #24871125 - 12/27/17 11:42 AM (3 years, 4 months ago)

You would have to be the one writing the tek. That's why these tools go to scientists who have a chance of being able to figure out how to do just what you're asking.

The sequencer will be cheap. Reagents, and disposable stuff will end up costing a fortune


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Re: Point mutation [Re: bodhisatta]
    #24872242 - 12/27/17 09:25 PM (3 years, 4 months ago)

A TEK, LOL!  Look how many years it took for them to sequence agaricus bisporus, I think that's the only one for now.  Maybe oyster and shiitake.  I would bet there's multiple groups working on finding out which genes do what now. 

I would just start by reading up on how the crispr button mushroom came about.  Maybe there's more literature out there but Idk.


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OfflineKrombopulosM
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Re: Point mutation [Re: drake89]
    #24872483 - 12/27/17 11:47 PM (3 years, 4 months ago)

Haha. Awesome info guys. Yea i was at the library today and i have to agree that this is not possible for me at home and takes a team of people who spent dozens of years in school in a real lab Lol. I'm gonna take small steps to get there tho. Thanks pros


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OfflineFishLevelMidnightS
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Re: Point mutation [Re: KrombopulosM]
    #24875647 - 12/29/17 10:44 AM (3 years, 4 months ago)

You can look up CRISPR/Cas9 technologies for inducing point mutations at a specific location. Even with a bunch of mycology/molecular biology/genomic background, it will take many many months to get an expected result.


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OfflineSuccubussasha
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Re: Point mutation [Re: FishLevelMidnight]
    #24917104 - 01/16/18 12:08 AM (3 years, 3 months ago)

I really don't think there is a CRISPR system that is used in mushrooms right now (each species needs a different plasmid to introduce the mutations).
I think the easiest system would be a chemical mutagenesis.
For instance, plant seeds are mutagenized with EMS. You put the seeds in a chemical for a certain amount of time (don't spill it on yourself). And then you just grow all the seeds and wait for ome to look different.
The amount of time that the seeds are in chemicals directly correlates to how many base mutations occur.(don't really need to sequence the genome if you find the phenotype you want)

Good luck.


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OfflineAlan RockefellerM
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Re: Point mutation [Re: KrombopulosM]
    #24917378 - 01/16/18 03:44 AM (3 years, 3 months ago)

Quote:

KrombopulosM said:
Does anyone have any good info on teks for point mutation?





CRISPR works.

Quote:

I feel like I'm gonna have to hunt down sequencing equipment pretty soon. They are under 3 grand now which is pretty affordable imo. Thanks for any help in this area.




More like $800 - 300 for a used PCR machine on ebay and 500 for pipettes / master mix / primers / tubes / gel box / Ethidium Bromide / Agarose / KOH / TRIS.


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OfflineSuccubussasha
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Re: Point mutation [Re: Alan Rockefeller] * 1
    #24917693 - 01/16/18 10:08 AM (3 years, 3 months ago)

Quote:

Alan Rockefeller said:
Quote:

KrombopulosM said:
Does anyone have any good info on teks for point mutation?





CRISPR works.

Quote:

I feel like I'm gonna have to hunt down sequencing equipment pretty soon. They are under 3 grand now which is pretty affordable imo. Thanks for any help in this area.




More like $800 - 300 for a used PCR machine on ebay and 500 for pipettes / master mix / primers / tubes / gel box / Ethidium Bromide / Agarose / KOH / TRIS.





To perform CRISPR you will need a plasmid vector to introduce your mutation, this vector will need to be cloned into e. coli to replicate it.
Then you will need to extract the e. coli DNA and PCR out the DNA you want (this will require primer design and ordering - the vain of my existence).
Then this DNA with the CRISPR/Cas9 vector can be introduced into a fungi, assuming that system exists (which I do not think it does).

CRISPR for every organism is different and requires a LOT of work, I spent 4 months trying to CRISPR a cyanobacteria and had no luck.

I highly suggest looking into chemical mutagenesis of the spores.
The mutations are random, but unless if you know the exact mutation that you want, each CRISPR edit will only give you the single mutation you selected for. With bulk mutagenesis you can mutagenize thousands of spores all at once, and it is unlikely any will have the same mutation.
You then just try to grow how you normally would, and look for one that has something different about it that you want to keep.


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OfflineFishLevelMidnightS
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Re: Point mutation [Re: Succubussasha] * 1
    #24917712 - 01/16/18 10:16 AM (3 years, 3 months ago)

Quote:

Succubussasha said:
Quote:

Alan Rockefeller said:
Quote:

KrombopulosM said:
Does anyone have any good info on teks for point mutation?





CRISPR works.

Quote:

I feel like I'm gonna have to hunt down sequencing equipment pretty soon. They are under 3 grand now which is pretty affordable imo. Thanks for any help in this area.




More like $800 - 300 for a used PCR machine on ebay and 500 for pipettes / master mix / primers / tubes / gel box / Ethidium Bromide / Agarose / KOH / TRIS.





To perform CRISPR you will need a plasmid vector to introduce your mutation, this vector will need to be cloned into e. coli to replicate it.
Then you will need to extract the e. coli DNA and PCR out the DNA you want (this will require primer design and ordering - the vain of my existence).
Then this DNA with the CRISPR/Cas9 vector can be introduced into a fungi, assuming that system exists (which I do not think it does).

CRISPR for every organism is different and requires a LOT of work, I spent 4 months trying to CRISPR a cyanobacteria and had no luck.

I highly suggest looking into chemical mutagenesis of the spores.
The mutations are random, but unless if you know the exact mutation that you want, each CRISPR edit will only give you the single mutation you selected for. With bulk mutagenesis you can mutagenize thousands of spores all at once, and it is unlikely any will have the same mutation.
You then just try to grow how you normally would, and look for one that has something different about it that you want to keep.




it might be different for different systems, but I made my sgRNA in a non-cloning method.

Gagnon et al 2014 even have a protocol for developing your own sgRNA in this method.

It was a lot easier for me and I could get sgRNA in a few days/work week once everything comes in.


Chemical mutagenesis is totally random in its effects, can be useful for phenotype hunting but if you want a specific phenotype and know a gene related, CRISPR is much faster.
If I want albino zebrafish it is a lot easier to disrupt tyrosinase via CRISPR than to hunt for an albino mutant. Plus, the albino I produce is only deficient in that one gene; a chemically or UV mutated zebrafish (spore, seed whatever) likely also has mutations in other regions of the genome which may have effects.


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OfflineSuccubussasha
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Re: Point mutation [Re: FishLevelMidnight]
    #24917740 - 01/16/18 10:36 AM (3 years, 3 months ago)

Quote:

fishermansjc said:
Quote:

Succubussasha said:
Quote:

Alan Rockefeller said:
Quote:

KrombopulosM said:
Does anyone have any good info on teks for point mutation?





CRISPR works.

Quote:

I feel like I'm gonna have to hunt down sequencing equipment pretty soon. They are under 3 grand now which is pretty affordable imo. Thanks for any help in this area.




More like $800 - 300 for a used PCR machine on ebay and 500 for pipettes / master mix / primers / tubes / gel box / Ethidium Bromide / Agarose / KOH / TRIS.





To perform CRISPR you will need a plasmid vector to introduce your mutation, this vector will need to be cloned into e. coli to replicate it.
Then you will need to extract the e. coli DNA and PCR out the DNA you want (this will require primer design and ordering - the vain of my existence).
Then this DNA with the CRISPR/Cas9 vector can be introduced into a fungi, assuming that system exists (which I do not think it does).

CRISPR for every organism is different and requires a LOT of work, I spent 4 months trying to CRISPR a cyanobacteria and had no luck.

I highly suggest looking into chemical mutagenesis of the spores.
The mutations are random, but unless if you know the exact mutation that you want, each CRISPR edit will only give you the single mutation you selected for. With bulk mutagenesis you can mutagenize thousands of spores all at once, and it is unlikely any will have the same mutation.
You then just try to grow how you normally would, and look for one that has something different about it that you want to keep.




it might be different for different systems, but I made my sgRNA in a non-cloning method.

Gagnon et al 2014 even have a protocol for developing your own sgRNA in this method.

It was a lot easier for me and I could get sgRNA in a few days/work week once everything comes in.


Chemical mutagenesis is totally random in its effects, can be useful for phenotype hunting but if you want a specific phenotype and know a gene related, CRISPR is much faster.
If I want albino zebrafish it is a lot easier to disrupt tyrosinase via CRISPR than to hunt for an albino mutant. Plus, the albino I produce is only deficient in that one gene; a chemically or UV mutated zebrafish (spore, seed whatever) likely also has mutations in other regions of the genome which may have effects.




Interesting!
But didn't you have to know the sequence you wanted to edit before using CRISPR?
The original post is vague but it sounded to me like the genome is probably unknown and the point mutation wanted isn't known either (in which case a mutagenesis screen sounds perfect).


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OfflineFishLevelMidnightS
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Re: Point mutation [Re: Succubussasha]
    #24917748 - 01/16/18 10:44 AM (3 years, 3 months ago)

You're absolutely right.

Sequencing the genome (or at least a transriptome) of the species in question would be the first step, then all the bioinformatics to assemble and annotate it.

Mutagenesis would be good for screening if it was a particular phenotype you were interested in, but you'd be hit also with weak genetics (unless you backcrossed).

Lots of time and space required! lol

I have a spore solution I exposed to ~40 min of UV light and plated on agar, I got growth and a few pins on the plate which is mostly dry. I'm curious what would turn up if I nocced up a jar with it...


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OfflineAlan RockefellerM
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Re: Point mutation [Re: FishLevelMidnight]
    #24918871 - 01/16/18 07:11 PM (3 years, 3 months ago)

Quote:

fishermansjc said:

Sequencing the genome (or at least a transriptome) of the species in question would be the first step, then all the bioinformatics to assemble and annotate it.






The best way to do that would probably to get a MinION.  They are only $1000, which is half what a company would charge you to sequence a full genome. 


https://nanoporetech.com/products/minion


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Re: Point mutation [Re: Alan Rockefeller]
    #24919517 - 01/17/18 12:00 AM (3 years, 3 months ago)

Wow!  When I was taking intro bio 7 years ago my professor told me it was a full time job running a sequencing machine that was a large million dollar device!  Now it's $1000 and the size of a phone!


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Re: Point mutation [Re: Alan Rockefeller]
    #24920438 - 01/17/18 12:51 PM (3 years, 3 months ago)

I saw gary lincoff talking about the MinIon on fb a while ago. The thing is that the flow cells which (as I understand it) are consumed with each read - cost $900. The starter kit includes two flow cells but after that you have to reinvest $900 everytime you want to sequence something.


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Offlinechibiabos
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Re: Point mutation [Re: FishLevelMidnight]
    #24921550 - 01/17/18 08:50 PM (3 years, 3 months ago)

Quote:

fishermansjc said:
If I want albino zebrafish it is a lot easier to disrupt tyrosinase via CRISPR than to hunt for an albino mutant. Plus, the albino I produce is only deficient in that one gene.



Wouldn't you still need to verify that your sgRNA doesn't bind to another site, to make that statement?  Or is there some minimum specificity that's required by the protocol for raising an sgRNA?


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Re: Point mutation [Re: chibiabos]
    #24921587 - 01/17/18 09:04 PM (3 years, 3 months ago)

sgRNA by design are fairly specific (similar to how primers work); there are predictors of locations that could be cut given any sgRNA target.

You can sequence the region of the genome where the sgRNA is designed to cut and look for evidence of NHEJ repair.

You can then sequence the regions predicted to have the potential for off target cutting. Alternatively you could use NGS to look for signatures of mutagenesis in the CRISPR induced individuals vs the wildtype reference genome.


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Offlinechibiabos
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Re: Point mutation [Re: FishLevelMidnight]
    #24921710 - 01/17/18 09:51 PM (3 years, 3 months ago)

Quote:

fishermansjc said:
sgRNA by design are fairly specific (similar to how primers work); there are predictors of locations that could be cut given any sgRNA target.



They're specific to complimentary sequences.  I'm not saying that CRISPR isn't way more specific than just punching holes through chromatin.  I'm just wondering about the extent to which you're able to say that your protocol only affected a particular region of DNA (in the sense that it's the singular region which your sgRNA was able to really bind to effectively) if you don't actually check.  It's probably not a huge concern if you're just trying to put some favorable traits into some mushrooms (or even for the vast bulk of wet-lab work), but I don't think that it's really a fair statement to make based on what amounts to an educated guess, however good of a guess it might be.  I'm not trying to begrudge bioinformatics, because bioinformatics is actually a really great thing, but it's pretty much just the computational end of some results of word math (which is probably not an actual term in academia).  Basically, here are some words (i.e. sequences of letters), here are some actions on those words, what are the results of those actions?


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Re: Point mutation [Re: chibiabos]
    #24921739 - 01/17/18 10:04 PM (3 years, 3 months ago)

You are right that off targets are a concern with CRISPR.

That word math is also what puts together a genome and it works well enough for us to fuck with it so...

There have been modifications to the CRISPR system that allow for increased specificity at the loss of available target sites.
I don’t feel like fishing up the references but essentially they mutated the Cas9 enzyme to be a nicase. Because it only cleaves one side, to get the double stranded break and subsequent mutagenesis via NHEJ, you need two sgRNA that are on opposite strands of DNA and within like 30-50 bp (less than 10 is preferred).
When these things come together you cut both strands and allow for NHEJ. The chances of finding another non target region of the genome where both sgRNA will bind close enough and on opposite strands is ridiculously low. The nicase alone causes damage that is repaired without the potential for mutagenesis, so off targets will not see a mutation. The paper was unable to find any signature of mutagenesis at non target locations with like 10X deep sequencing.


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Offlinechibiabos
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Re: Point mutation [Re: FishLevelMidnight]
    #24921809 - 01/17/18 10:34 PM (3 years, 3 months ago)

Quote:

fishermansjc said:
You are right that off targets are a concern with CRISPR.

That word math is also what puts together a genome and it works well enough for us to fuck with it so...




Nope, that would be electromagnetism.  What I'm talking about is more related to things like counting theorems and symmetry groups.  The word math is just a set of axioms that will let you describe the results of some collection of actions on words.  You can absolutely come up with a word that corresponds to any region of any polymer, but that doesn't mean that the polymer and its image via that correspondence are the same thing.  One is a part of a molecule and one is an abstract concept which is abscribed some particular properties (like order, maybe even a basis and so on).  There is sort of a topical relationship between the two, but the latter is realized in a way that doesn't resemble the former at all (in any other sense than them corresponding to that same word).


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