New & Noteworthy

Knock out YME1, Luke

April 20, 2017

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In the original Star Wars, Luke destroys the Death Star with a precise strike of proton torpedoes down a small thermal exhaust port. For him it was as easy as bullseyeing “womp rats in my T-16 back home.”

Luke and the rest of the Rebel Alliance learned of this engineered fatal flaw from Jyn and her friends in the prequel Rogue One. With this information the Rebel Alliance was able to keep the rebellion alive long enough to finally bring down the Empire by the end of Return of the Jedi.

It turns out that our friend Saccharomyces cerevisiae has taught us about a fatal flaw in mitochondria. Like proton torpedoes in an exhaust port, when the gene YME1 is inactivated, mitochondria become unstable. But instead of bits of Death Star raining down on nearby planets, mitochondrial DNA (mtDNA) is released into the cytoplasm.

Sometimes this mtDNA can end up in the nucleus and find its way into nuclear DNA. And if the conclusions of a new study in Genome Medicine by Srinivasainagendra and coworkers turns out to be right, this numtogenesis (as the authors call this process) can have profound consequences when it happens in people. Their data suggests that it might lead to cancer or possibly cause cancers to spread.

These researchers searched through whole genomes of colon adenocarcinoma patients and found that these cancer cells had 4.2-fold more mtDNA insertions compared to noncancerous cells from the same patient. They also found that patients with more of these insertions tended to do worse (although the sample sizes were too small to say this definitively).

Why is this happening in the cancer cells? What has caused the mitochondria to give up their DNA?

Srinivasainagendra and coworkers turned to previous work that had been done on the YME1 gene in the yeast S. cerevisiae to find one possible reason. YME1 had been shown to be an important suppressor mtDNA migration to the nucleus. Perhaps this was true in mammalian cells as well.

A search through the genomes of cancers suggested that this seemed to be the case. Around 16% of the colorectal tumors they looked at had a mutated YME1L1 gene, the human homologue of YME1. And mutated YME1L1 genes were found in other tumors as well.

They used CRISPR/Cas9 to directly test the effects of knocking out YME1L1 in the breast cancer cell line MCF-7. The knock out cells had a 4-fold increase in the amount mtDNA in the nuclear fraction compared to cells that still had working YME1L1.

As a final experiment, they used a yeast strain, yme1-1, in which YME1 function was inactivated, to show that the human homologue, YME1L1, could suppress the migration of mtDNA to the nucleus.

This yme1-1 strain has a TRP1 gene encoded in the mtDNA instead of the nucleus. Since the gene cannot be read by the mitochondrial transcription machinery, the only way this yeast strain can survive in the absence of tryptophan is if the TRP1 gene moves from the mitochondrion to the nucleus. 

In their experiment, with vector alone, they got around 1000 TRP+ colonies with yme1-1. When they added back yeast YME1, this number dropped to less than 50 compared to the 100 or so they got when they added the human homologue, YME1L1. So YME1L1 can suppress mtDNA migration to the nucleus.  

Given that YME1L1 was mutated in just a subset of the cancers, it is unlikely that it is the only player in the mtDNA these authors found in the nuclei of cancer cells. But it does look like it is one way this can happen.

And it would have been very hard to fish out the human gene without the critical work that had been done in yeast previously. Yeast shows us the way again. #APOYG

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: cancer, colon cancer, CRISPR/Cas9, MCF-7, mtDNA, nuMt, nuMtogenesis, YME1, YME1L1

Control (Apple) F for Genetics

May 18, 2016

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A long time ago, through the mists of time, finding a phrase or a passage in a document was hard. If you didn’t have the foresight to highlight it, you were stuck.

You could reread the whole document but that might take way too much time. Or you could narrow down where to look by remembering the chapter it was in and rereading just that.

Nowadays of course, finding that phrase or passage is trivial in a Google or Word doc. You just use the find function!

Finding the genetic variant responsible for a given trait is still, in many ways, in the age of the typewriter. A genetic association study can find a part of the DNA responsible for that trait but homing in on the exact variant is terribly time consuming and labor-intensive.

Enter that wonder of a gene editing tool, CRISPR.

In a new study in Science, Sadhu and coworkers have essentially turned CRISPR into a find function for genetic association studies. They use CRISPR’s double stranded DNA cutting ability and mitotic recombination in a heterozygote yeast strain to blanket a region of DNA with crossover events.

Sifting through the results let them find the actual amino acid change responsible for manganese sensitivity with much less effort than would have been required with the old method. They only needed to use 358 lines instead of the 7,500 or so they predict they would have had to go through without good old CRISPR.

And think how much time might be saved with a more complicated beast! Given its short generation time and relatively small genome, yeast is like a magazine article. A human is more like War and Peace. This new system might make linking human traits/diseases to the causative SNP much simpler.

The usual way to link a trait to the DNA that causes it is to see what known traits tend to get passed down with it. If you know trait A is at position X on chromosome Y and the trait you are interested in, trait B, is usually passed down with A, then trait A and trait B are close to each other (this is called linkage mapping). This is the easy part.

The hard part is getting from A to B. To get finer mapping, you need to rely on the recombination that happens during meiosis. This is the step where this new study can help.

Instead of relying on the slow, natural process of meiotic recombination, these authors use the CRISPR-Cas9 system to jump start mitotic recombination.

They start off with a heterozygote in which one of the parent strains has a trait and the second does not. They chose the lab strain, BY, and the vineyard strain, RM, as the parents.

Next they designed 95 guide RNAs that would direct Cas9 to 95 different spots along the left arm of chromosome 7. Sadhu and coworkers targeted sites that were heterozygous in the two strains.

Once Cas9 cuts the DNA, the cell uses homologous recombination to repair the cut using the DNA on the other chromosome in the pair as the template. This results in a loss of heterozygosity (LOH) which can reveal recessive traits.

They picked 384 lines, approximately 4 from each cutting event and found that 95% of these had undergone LOH with hardly any off-target effects.

They next measured the growth of these strains in 12 different conditions and found that one trait, growth on 10 mM manganese sulfate could work for their purposes. Using the more traditional approach with 768 segregants obtained through meiotic recombination, they were able to narrow it down to an overlapping piece of DNA of 3,900 bases. However, using CRISPR-Cas9, they were able to narrow down the location of the variant to a 2,900 base pair stretch of DNA. Score one for the new method!

Next they did finer mapping by targeting the 2,900 bases they had identified with CRISPR-Cas9. Even though they only used three guide RNAs that targeted three locations, they were able to look at many more places on the DNA than this because the length of DNA repaired around the cut varies a bit between strains.

They isolated 358 lines of which 46 or 13.1% had a recombination event in the 2,900 bases they were interested in. Only 0.7% of segregants obtained the old way were in the right place. Score another one for the new method!

After measuring how well these 46 lines grew in manganese sulfate, the researchers were able to narrow the search to a single polymorphism that resulted in an amino acid change in the PMR1 gene. A perfectly reasonable result, given that Pmr1p is a manganese transporter. They then showed that this variant, pmr1-F548L, did indeed make a strain sensitive to manganese all on its own.

By targeting recombination events to regions of interest, these authors have provided proof of principle for a technique that should make connecting traits (phenotype) with DNA variants (genotype) much easier than it is currently. Behold yet again the awesome power of yeast genetics (#APOYG)!

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: CRISPR/Cas9, genotype, mapping, phenotype

Keeping Gen(i)e Drives in Their Lamps

December 02, 2015

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Everyone knows about genies. They have almost infinite power, can grant you three wishes, and are kept under control by the owner of their lamp.

And as we saw in Disney’s Aladdin, it is a good thing that the lamp is around! When the evil sorcerer Jafar was given the powers of a genie, he began to take over the world. Until, that is, Aladdin forced him back into his lamp where he could be kept under control.

In the last few years, scientists have come up with their own genies. While not as powerful as the “real” ones, these gene drives can still pack quite a punch. And maybe even grant us a few wishes.

Gene drives can force genes to spread quickly through a population whether those genes are good for a species or not. This means we might be able, for example, to force a “bad” gene to spread through the mosquitoes that transmit malaria. By causing the mosquito population to crash, our wish to save hundreds of thousands of lives each year would be granted!

But just like a genie, we need to keep gene drives under control. We do not want something that overrides natural selection to escape and wreak havoc with ecosystems.

Which is where, as usual, our friend yeast can help! In a new study out in Nature Biotechnology, DiCarlo and colleagues use yeast to test two different strategies to make gene drives safe enough to use. And, they argue, safe enough to research.

Gene drives are based on the idea of homing endonucleases. Basically, if a gene associated with a gene drive is on just one of the two chromosomes in a pair, the gene drive will copy and insert the gene into the other chromosome through a precise DNA cut.

Now both chromosomes end up with a copy of the gene. Which of course means all of the offspring will get the altered gene too. This copying will happen generation after generation until the new gene has swept through the population.

The idea for gene drives has been around since 2003 but really only became practical with the discovery of the CRISPR/Cas9 system. This genome editing tool, which is ludicrously simple to program to target most any DNA sequence, allows scientists to create most any gene drive they want.

The CRISPR/Cas9 system has two parts. One part is the guide RNA which leads the second part, the endonuclease Cas9, to the right spot in the genome to cut. What makes the system so powerful is that you just need to make a different guide RNA to target different sequences in the genome.

One easy way to help control a gene drive is to keep these two parts separate. Do not have the guide RNA and the Cas9 on the same piece of DNA. Then, if one part were to escape, it couldn’t do anything on its own.

This is of course easy to do in yeast. Just integrate one part into a chromosome and keep the second part on a plasmid.

This is just what DiCarlo and coworkers did. And they showed that this separation can be very effective.

They integrated a guide RNA into the ADE2 gene of a haploid yeast to create a gene drive designed to disrupt ADE2. As expected, this strain produced red colonies on adenine limiting media.

They next mated this strain to a wild type haploid. All of the resulting diploids were cream colored. This is what would be expected as both copies of ADE2 need to be disrupted to see red colonies in a diploid.

When these diploids were sporulated, the researchers got the expected 2:2 ratio of red to cream colored haploids. This all changed when they introduced a Cas9 containing plasmid into the experiment.

In the presence of Cas9, more than 99% of the resulting diploids were red. And when sporulated, these diploids produced all red haploid colonies.

The two parts of CRISPR/Cas9 together drove the disrupted ADE2 through the population. But importantly, just having the guide RNA integrated into ADE2 had no effect on how the two alleles were passed down. Once one part is removed, the gene drive stalls out.

The same system also worked when the ADE2 gene drive included the URA3 gene so that URA3 spread through the population as well. It also worked when the essential gene ABD1 was targeted.

And genetic background did not significantly affect how well this ADE2 gene drive worked. When they mated their haploid to six different strains of yeast they saw no loss in efficiency.

So separating the two parts of the gene drive is a pretty good failsafe. But of course nothing is perfect.

Ideally we need some way to shut the system down if all of our safety features fail. We want to be able to get rid of Jafar and the lamp entirely if possible.

DiCarlo and coworkers showed that they could create a gene drive that could overwrite and correct the ADE2 they had disrupted with the guide RNA. This new gene drive targeted a synthetic sequence in the original gene which means that it would only affect altered yeast. So even if things go awry, we may be able to erase the changes we made.

These two strategies should help keep gene drives in check both in the wild and the lab. But of course, again, it is important to keep in mind that nothing is foolproof.

At the end of Aladdin, they buried Jafar and his lamp deep in the desert to keep him from causing any more trouble. But his lamp was found and Jafar reemerged to wreak havoc in the second Aladdin movie, reminding us that we must be very careful when unleashing powerful forces.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: CRISPR/Cas9, gene drives

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