Second Generation GMO tools: Talens and Crispr

New GE tools are being created, called "Talens" and "Crispr". Those are much more precise than the "traditional" GE tool based on Agrobacterium plasmids. The old way could only encourage added DNA to splice into the plant genome "anywhere" - any location on any chromosome. Unless the added DNA included a promoter region and other regulatory regions, the descired genes would have to get lucky to be expressed or controlled even if they did splice in "somewhere".

And the plasmids usually carried along at least some excess baggage, some of which might have been not well characterized.

Then old Agrobacterium plasmid method was a poorly-aimed shotgun. It was used to blast some genes into a tissue sample willy-nilly, and then they used tissue culture to pull out some of the "random hits" and see what they got. The tendency was to lob in genes from other species to get a big, obvious change.

"Talens" and "Crispr" splice DNA into exactly known locations (an exact DNA sequence on one plant chromosome). Thus they will inherit the plant's existing control mechanism for that gene's expression.

I think the two new techniques tend to rely on known ("sequenced") segments of plant DNA or specific changes (i.e. intentional, controlled mutations). Modifying an existing gene is just like selecting for a different allele of that gene - like selecting for blue eyes instead of brown. It's not like adding "eyeball genes" to a tomato!

They can be used to modify an existing plant gene to a different version (different allele, perhaps a new, "designed" version of the gene). For example, you could use them to change just a few base pairs to alter a few amino acids in one enzyme.

"Talens" and "Crispr" have even been used extremely gently: as a "fast way to make a cross" when breeders wanted just one gene changed to a version found in (for example) a wild version of the same species. If conventional breeding was used, the cross would take many plant generations to first pull in the wanted version of the gene (wild allele) and then breed the result back to the commercially valuable other traits. With "Talens" or "Crispr", you change only the part of the gene that you want to change, nothing else.

Either of those should be a much less scary change than slamming in entirely different genes from entirely different organisms.

Talens uses engineered proteins to recognize the desired stretch of DNA to modify or "crossover".

Crispr uses RNA to recognize the desired site.

That's the good news: they are more precise and subtle tools that will let plant geneticists make smaller changes to have better-controlled effects.

Good or bad news: when the changes are not transgenic, US regulators will let field trials occur with less hoop-jumping than they required for the transgenic genetic changes. That would be good news for poor countries wishing someone would put genes THEY need into some crops. If the changes made are designed or invented changes as opposed to DNA sequences borrowed from wild versions of the same species, they are still "new" DNA being released into fields.

Other mixed news: at least one company is selling tool kits and start-up services for the Talens and Crispr tools. The new tools are not only more powerful and their products under less regulatory scrutiny, but they don't seem to be very expensive. That may weaken the monopoly or oligopoly that a tiny number of huge companies have (good news). It will also let many more small companies and national laboratories into the game (good or bad news).

kits & reagents for do-it-yourself GE
http://www.genecopoeia.com/about/overview/

"Precise Genome Editing Tools"
http://www.genecopoeia.com/product/genome-editing/?gclid=CJu8y5OhorwCFYhsfgodDGMAAA

TALEN kits starting at $595.

" Genome-CRISP™ CRISPR-Cas9 products and services

Fast CRISPR-Cas9 sgRNA design and cloning services. Validation and other related services also available for simple and fast targeted genomic editing.

Starting $345"


Last, a kit that is good news for anyone with a genetic disease and medical researchers who want to cur such diseases. Scroll down to the bottom of the page from the second link above.

" TALEN- or CRISPR-mediated gene targeting kits designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site
in human chromosome 19"

I am so torn about this issue. It would be great for India or Africa or Haiti to have more food with fewer seeds (+) but that shouldn't prevent them from saving seeds nor force the farmers (poor to begin with) to be in hock to Monsanto forever (-). Maybe some kid at MIT will buy a kit and figure out how to grow corn w/o pesticides (+). Maybe some kid at MIT will figure out how to use the kit to clone Adolf Hitler or Calvin Coolidge (-). I hope that if gene altering is open-source, if you will, people like Monsanto will stop making ungodly profits out of other people's misfortune.

It should be good news for people (like me) who are professionally ill, by which I mean I never go more than an hour from away from a hospital or wheelchair repair store and my medical expenses are ALWAYS more than 7% of my income.

Adolf Hitler and...Calvin Coolidge????

In recent years, Monsanto's stock price has lagged the S&P 500, not exactly an indicator of "ungodly profits".

Rick--Interesting information; not sure what to make of it all. I can say I won't be buying a TALEN or a CRISPR kit.

>> but that shouldn't prevent them from saving seeds

My hope is that it does enable more organizations to create and release new varieties of NON-transgenic crops that don't frighten as many people.

The expense of developing and releasing GE crops is currently almost entirely the testing process. Many hundreds of varieties are developed far enough to apply for testing permits that never go into production.

If the cost to develop a variety is much less, the ability of not-for-profit groups to do it increases. Or, if a corporation does it, they might be able to recoup development and testing costs while allowing third-world farmers to save their own seed crops.

Then I hope that some of those new varieties are targeted at low-input farming (dry and marginal soils, hotter climate, saline water, less fertilizer, less and fewer pesticides, less mechanized cultivation). As the population continues to rise and climate change brings more frequent weather extremes, increasing yields and bringing sustainable crop production to marginal land and climates may help avert or reduce widespread famines.

More efficient utilization of low-rainfall areas, alone, could reduce human misery a lot.

Some numbers for transgenic GE crops for which there were US permits and notifications for testing but no commercial release:

863 potato varieties
461 wheat varieties
286 rice varieties

(source: Jan 2014 MIT Technology Review)

Hasn't there been concern that we are losing varieties due to this industrial agricultural moncropping etc. I mean now we can invent new varieties everyday both by GMO and non GMO methods...good old plant breeding! Now there is more variety diversity as there ever has been. Farmers have more choices than they ever have. And how about in the store...remember shopping for apples 20 years ago? You had red delicious, red delicious and red delicious and maybe a granny smith. Or how about lettuce...iceburg head lettuce and that was about it...

"...people like Monsanto will stop making ungodly profits out of other people's misfortune." I agree Monsanto hasnt been doing so well...did anyone ever realize that glyphosate, the active in greadient in Roundup is off patent and there is alot of generic competition. And whatever profits they do make do not come out of anyones misfortune. Profits usually come from a good idea and a lot of hard work.

Of course there are many who have misfortunes...all of us do...but they are not generally the result of somebody who is successful. For example I cant blame my bad grade on an exam because somebody had a good grade on theirs. I can only attribue it to my efforts or lack there of...or not preparing or even just plain bad luck. Maybe not having a good idea...or not having good health or being in the right place at the right time...or not taking a risk.

There is not a finite amount of success...and the more success one person has does not limit the success of others. Success is infinite.

I beleive in equality of opportunity. But I do not beleive in equality of success or results. Since we are all individuals and all different we each will have varying levels of success.

Drobarr,

I agree completely with you, but there are many people in this world that do not. An ex son in law of mine told me 30 years ago, that my generation had used up all the opportunity and none was left for people of his generation, so they had no choice but to spend their lives laboring in factories.

And of course, there has been more progess in the last 30 years than ever before.

Ernie

Ernie,

While your ex son in law may have been sort of right...that it does seem like opportunity is in shorter supply...but not because of anyones generation or that it was used up.

The more society in general favors equality of success or results, the less equality of opportunity there is to be above average or to truly excel. This is because the motivation and the reward is eliminated when results are equalized. Risk takers have to be rewarded or nobody will take the risks. Why take the risk if the result is the same or similar to not taking the risk or doing the work, or coming up with the idea.

But there is still plenty of opportunity out there...but if you dont believe that there is...you will never find it.

Sorry to get off track here...just get irritated when people dis good ideas, hard work and success! If anyone should know that it is a gardener! You havest what you put into it.

Drobarr,

Since we are discussing the Risk Monsanto has taken to explore the Opportunities they see in GE and GMO, i think we are on track here.

I have no way of judging whether there is more or less opportunity now, but i recall how negative everyone i talked to was, about the risk of Failure, back when i was in my twenties. I had a difficult time borrowing the last $600.00 i needed for a Workmens Insurance Policy, as everyone was sure there was no opportunity to ever make enough to pay it back.

Then, almost forty years later, when i was entering a completely different industry, I heard the same negative opinions, which were wrong again. So there has always been a not of discouragement out there. keeping people from trying.

So maybe the opportunities are just obscured by stronger Risk Aversion, so companies like Monsanto should be applauded for having the courage to risk so much to try to do something that will help us all in a brand new field, if it succeeds.

The naysayers do not seem to realize that if we do not increase food production to keep up with increasing population, that Starvation will kill a lot more peole than a few mistakes that may be made while seeking progress.

Ernie

Ernie,

"The naysayers do not seem to realize that if we do not increase food production to keep up with increasing population, that Starvation will kill a lot more peole than a few mistakes that may be made while seeking progress."

Very well said! Nothing is perfect...but its doing the best we can with what we have. You can always find imperfections with progress. But often the progress is a step better than what was done before.

For my money, I think this subject is inherently political. Nothing at all wrong with addressing the political side of the issue.

Did you ever wonder why it's only those big, evil, profit-grubbing corporations that are in the business of producing genetically modified foods? It's because, unlike any other form of genetic modification (including chemical and nuclear), molecular genetic modification is regulated by the government. It's very expensive and time consuming—small businesses just can't compete.

One of the great puzzles to me is the fact that people who want more government regulation are those who also despise big business. I'm not arguing against regulation, but it does come at a hefty cost and it often precludes small businesses from entering many fields. In my town, mom and pop restaurants are slowly giving way to big chains--Chile's, Olive Garden, etc. An acquaintance who is in the restaurant biz--and who's restaurant just went under-- tells me she thinks more mom and pops will soon be going away as the city begins to require enhanced grease collection systems; systems that run over $50,000 per facility. So, we eat at big chains and the restaurant scene in a small Arizona town is slowly beginning to be identical to one in New Jersey, Alabama, Alaska, or anywhere else.

Rick's initial post here suggests that the technology of genetic modification itself is not, and probably hasn't been for a while, very costly. It's the costs of compliance that are huge and the risk of failure is huge too. Imagine spending hundreds of millions and having the tested product be rejected. Jane and John Doe just can't do that.

Anyway, them's my thoughts.

Willy,

Your thoughts exactly match what I have been preaching since 1972. But i would like to add one fact to what you say. Actually, the big corporations do not pay for the $50,000.00 Grease Catchers regulations require nor any of the other expenses. The simply finance them and eventually pass the cost on to the customers.

Most of what i post here is based on personal experience, and like your friend that closed his restaurant, back in 1970,, when the regulations required that i install a vaccuum cleaner that cost half as much as my Asphalt Plant was worth, I could not afford it, and shut my plant down. It did not matter that the dust from the plant was being scrubbed with water spray and not leaving my property, the Bag House, which was as big as a Highway Truck Trailer had to be installed.

But the rest of my company survived, and by the time they required vacuum cleaners for the Rock Crushers, i had learned how the big corporations were handling it, so i just financed the required environmental equipment and passed it on to the consumers as a business expense.

So, now, when the taxpayers have their streets paved or buy some concrete that has rock in it, they are paying an extra 50% of what the actual rockcrusher cost, just to catch dust that in most cases would never leave the crushing sites. It is regulations like this, that in a large part of the time are not necessary, that has raised the price of everything.

I paid $32,000 for a new five bedroom house in a nice subdivision in So. CA. in 1972. Now, those houses sell for several hundred thousand, so many young families cannot afford them, and the major reason for the increased price is environmental regulations that reach in to every part of that house, from the developer having to protect lizards and dragon flies, to manufacturers of every component.

IF THE CONSUMERS THAT ARE PAYING FOR EVERY PENNY OF THIS WERE AWARE OF IT, they would demand some common sense be applied to both the regulations and the enforcement of the rules. But as long as they believe the Politicians that tell them the EVIL RICH CORPORATIONS PAY FOR SO IT IS FREE, things are only going to get worse.

Of course i believe in clean water for domestic purposes, and clean air where there are people breathing it, but the regulations go way beyond that,

Ernie

You are right about people ultimately paying, be it through taxes or the price of a product. I made a similar comment somewhere in the GMO thread. The trouble with raising prices to cover these costs is that the small restaurant has to raise prices enough that maybe consumers leave anyway. Your $7 or $8 burger is now $10 or $11--why not just go to Applebee's. And it can be difficult for a small business to get a large enough loan from a bank that wants pretty high confidence of repayment.

For sure, Competition is always tough, and the more you need financing, the tougher it is to get the loan.

Ernie

I'll bow out of the political part of this thread, since it doesn't seem particularly focused on the specifics of GMOs, and I have yet to see anyone change any political opinion whatsoever as a result of an online conversation.

>> It's the costs of compliance that are huge and the risk of failure is huge too. Imagine spending hundreds of millions and having the tested product be rejected. Jane and John Doe just can't do that.

That is partly what I was saying in this thread, except that it's more true of the "first generation" GE tools (Agrobacterium plasmids and so called "biolistics" - just blasting DNA particles mechanically into plant tissues and relying on some getting into a nucleus and randomly crossing over into the cells' DNA).

Pro-technology people and anti-government people will probably be glad that the newer GE tools are specific enough and powerful enough that there is less motivation to go to the brute-force method of using transgenic DNA to get a desired effect.

Now that we have sequenced plant genomes and cheap fast sequencing tools, and Cripsr and Talens to do pinpoint insertions and crossovers, "controlled mutations" and "instant breeding" are possible. Genes from wild cousins can be imported more cheaply and faster than could be done with conventional breeding.

I was trying to make the point that the new tools also greatly reduce the regulatory burden of releasing a new crop variant.

Therefor, Jane and John Doe and Habib and Bohlale (or an under-funded private or government lab) an develop and release a new crop variety without spending a cubic yard of money OR frightening many people by slinging transgenic DNA around WITH the plasmid zipper that lets it pop in and out of genomes.

Rick--I do not understand why these particular technologies will allow people to manipulate genes on a molecular level, yet not "scare" people who are anti-GMO. My suspicion is that any form of manipulation is rejected by the hard core anti-GMO crowd. I would think that the futures of these technologies will likely be as tightly controlled as GMO research is currently. I may well be missing something here, because once the discussion gets heavy into the technology itself, my understanding of biology is poor and I get overwhelmed by the terminology. I am thankful for your reasoned and detailed explanations throughout these threads.

I agree with you on one level about disliking the "political" aspects of these discussions, but I do think politics and GMO are so intertwined that it's hard to keep them separate. Profits have been at least tangentially mentioned from the beginning of these threads, and, at least for some, the "fact" of "ungodly profits" becomes a major reason to oppose GMOs. Soon, we're worried about Hitler and equate him to Calvin Coolidge. Science takes a back seat to hysteria.

This message was edited Feb 18, 2014 3:14 PM

The level of regulations in any given industry has a direct affect on the size of those businesses in those industries. As regulations increase, the number of companies able to comply with those regulations decreases. The size of the remainig business tend to increase.

Big oil, big drug company, big pesticide companies etc are all big because the government has created regulatory environments that has favored larger companies. As companies become larger and fewer, competition is reduced and the consumer not only is stuck with the bill for the regulations but the bill for higher prices due to less competition.

I am not against big companies or regulations. But there should be a proper balance. I dont know where that balance is. Many would argue the current regulations are to strict, many argue they are not strict enough. And a few think they are good where they are.

In many industries we have regulated them to death which is why they are no longer here and have moved elsewhere.

As was pointed out other regulations have made home ownership impossible to many people...How about a country that won the space race by sending multiple people to the moon and back and now we depend on the loser of the cold war to get our astronauts into orbit. Regulations can protect, but they can also destroy.

I was dumbfounded when I first read about "biolistics" in Pollan's "In Defense of Food", though I don't think he used that term. What a contrast to the stereotype of high tech. Anyway, that raises a big question in my mind. Does the "bullet" need to put whatever the desired gene is in just the right spot to "link up" or can the gene be deposited in any one of several spots or can the gene deposit be in many number of spots and "link up"? Put another way, does each single shot produces a few, or many, successes? Or maybe it takes many shots to get a single success? Maybe a better way to ask is if the genome of one GMO stalk of corn (or whatever) identical with respect to individual gene locations to any other stalk of the same type of GMO corn, or is there variation from one stalk to the next with all equally expressing the same intended trait? My apologies for the crude terminology. I hope the question is intelligible.

Agreed, but I would add that it takes a back seat to financial interests, deceit, political dogma on both sides, and desires to be re-elected or advance a partisan agenda on either side. Poor Science! Nothing on her side but Nature and truth.


>> I am not against big companies or regulations. But there should be a proper balance. I dont know where that balance is.

>> Regulations can protect, but they can also destroy.

Drobarr, I agree with everything you said in that post.

Willy, great points! And it’s a very good question. I understand what you're asking and the answer is typically depressing for scientists. It might take dozens of shots to get one usable cell, and then they have to clone that cell to get enough seedlings to test.

>> biolistics

Did you hear that the earliest "gene guns" were modified Crossman air pistols?! I'm sure that some lab team had tried many expensive methods and they all stunk. I've read that researchers had been trying for years and found no good way to "transform" plant cells. Then some lab assistant probably brought in his pellet gun and they had a good laugh but tried it anyway. Then found that it worked better than the other methods.

(They have fancier gene guns now, but I forget the mechanisms. Something involving powdered tungsten.)

>> or is there variation from one stalk to the next

No, they select one or a few promising "transformed" plant cells and grow them each one out into many identical (cloned) seedlings. Then they test those, and usually do some breeding (conventional breeding).

>> Does the "bullet" need to put whatever the desired gene is in just the right spot to "link up" or can the gene be deposited in any one of several spots or can the gene deposit be in many number of spots and "link up"?

>> Put another way, does each single shot produces a few, or many, successes?

The newer techniques (Talens and Crispr) make the DNA changes in a precise, predetermined spot. That is their advantage. The old ways (biolistics AND the Agrobacterium plasmid) were like shotguns: they added new DNA randomly, wherever it happened to attach to a chromosome.

So the genengineers have to FIND some cells where the DNA succeeded in slipping into the plant DNA. Then each cell would be tested to see if it was what they wanted.

Let's make up some numbers for convenience. Suppose they started with a sample of plant tissue with 1,000,000 plant cells. They would grind it up (or dissolve it somehow - I don't know) into a slurry of single cells, and let's say 100,000 single plant cells made it to that stage.

They would shoot the 100,000 cells with plasmids or micro-pellets containing a continuous piece of DNA containing several parts.

1. the new gene(s) they WANT to add
2. the plasmid or other DNA sequence that can help "splice" the forign DNA into a plant chromosome
3. a "marker" gene for "antibiotic" resistance - making the plant cell resist a certain antibiotic
4. odds and ends of unwanted random DNA carried along from when the parts were spliced together.

Of the 100,000 cells, say 30,000 got the DNA injected into the cell.

Of the 30,000 cells that were "hit", say the DNA penetrated the nucleus of 5,000 cells.

Of the 5,000 nuclei with foreign DNA inside, say 50 of them had the DNA succeed in splicing its way into a chromosome. These were "transformed".

Of those 50 transformed cells, say 5 spliced into a spot where they became active or were "expressed".

Next the biologists did the easy part: finding the 5 successful plant cells that had been transformed and had their new DNA expressed. They squirted the antibiotic into the tube or petri dish containing 1,999,995 unwanted cells plus 5 interesting cells, and stuck it into a tissue culture incubator.

The 1,999,995 unwanted cells die. The 5 other plant cells are the ones that got the antibiotic resistance gene. If they got that, they also got the transgenic gene that the genetic engineers originally wanted to insert, because the transforming DNA was one big piece and the two genes "associate together".

Each of the five cells would live and multiply into little blobs that then could be coaxed into 5 micro-seedlings, then seedlings, then plants.

I think instead each blob is isolated and dissolved again and cloned into 100s or 1,000s of identical seedlings to be tested or bred further.

So with those numbers, one "experiment" is likely to produce a few cells with a genetic change. The breeding stock has to be created from that one cell. I don't know how many hundreds or thousands have to be tested before they find one that is worth developing farther.

I think my numbers were too optimistic by 100X or 1,000X for the gene gun. I suspect that they have to shoot a lot of test tubes with gene guns just to get one cell that was transformed the way they hoped. The Agrobacterium plasmid was the answer to their dreams: it was pretty good at inserting DNA, and they just had to hope it would be expressed.

I suspect that each time one part of the lab comes up with a "recipe" for a genetic change they would like to test, the part of the lab that carries out the above process tries dozens of times to produce a few viable transformed cells that can be cloned into a tiny test crop. Then they probably have to go back and try different recipes, like different transgene alleles, or maybe they also played with different promoter regions back then.

Science (or in this case engineering) is expensive and slow and takes incredible patience. I quit research and went into software because I didn't have enough patience.