New & Noteworthy

Trouble with Triplets

April 06, 2018

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This Research Spotlight also comes as a video animation. Be sure to check it out!

In the Star Wars movie Attack of the Clones, Padme and Anakin end up fighting Genosians on an automated assembly line. Padme manages to survive because the assembly line is set up in an ordered and predictable way. She knows when to jump, when to pause and so on to survive. If there was more chaos in the line, she might have been killed and then there’d be no Luke or Leia!

If the Genosian assembly line weren’t predictable, Padme might’ve ended up a pancake.

The same kind of thing can happen when cells read genes. An enzyme called RNA Polymerase II (Pol II) slides along the gene, spewing out a long string of messenger RNA behind it.

Just like the assembly line on Genosis, the gene is set up in an ordered and predictable way. Nucleosomes (barrel-shaped clusters of proteins) are spaced along the gene’s DNA to help keep it from getting tangled up. But when Pol II comes hustling along, the nucleosomes need to be carefully removed in front and then replaced after it passes.

Similar to the plight of poor Padme getting hurt if the assembly line were not predictable, genes can likewise get damaged if the nucleosome removal/replacement process goes haywire. In Star Wars, the result of a defective assembly line is damaged droids and TIE fighters. In cells, the result is a changed assembly line—the gene can end up longer! This change can be harmful for both the gene and the cell.

In a new study in GENETICS, Koch and coworkers use our favorite beast, the yeast Saccharomyces cerevisiae, to show that the ISW1 gene is a key player in making sure that the nucleosomes get back on DNA after being taken off. When yeast lack the ISW1 gene, nucleosomes don’t always return after being removed to make way for Pol II. And for some genes, this spells even more trouble.

The genes that have the most problems are those that have something called triplet repeats. Basically, this means the same 3 bases (e.g. CAG) are repeated many times in a row in the gene.

Using PCR, Koch and coworkers showed that a gene with triplet repeats ended up with more of them if the ISW1 protein (Isw1p) wasn’t there to shepherd the nucleosomes properly. And too many repeats in a gene can be harmful–not just to the yeast, but to us as well.

In fact, Triplet Repeat Expansion Disorders happen when the number of repeats increases from one generation to the next.  These diseases are some of the most devastating ones around.

They mostly cause slow but steady degeneration of nerve and brain cells, and the cruel symptoms (loss of body movement control, dementia) often only show up in mid-life. The most well-known is probably Huntington’s disease, which killed folk-singer Woody Guthrie.

So understanding how Isw1p helps keep the Pol II assembly line running smoothly in yeast might help us understand how triplet repeat expansion happens in humans, and may eventually give us ideas how to keep it from happening in the first place. This is especially likely because humans have proteins that are similar to Isw1p and probably do something similar.

To determine how Isw1p regulates nucleosome reassembly, Koch and coworkers used Southern blots to show that yeast that lacked Isw1p couldn’t replace their nucleosomes after Pol II had passed as efficiently as yeast that had the protein. This means that when cells are missing the ISW1 gene, a long stretch of the DNA is left bare after Pol II has passed by.

This stretch of nucleosome-free DNA can end up forming new structures called hairpins that can cause cells to send in DNA repair machinery to deal with it. Unfortunately this machinery isn’t always that great at fixing the DNA. Like the Three Stooges adding more pipe to try to fix a leak, the cell can end up adding more DNA to deal with the hairpin.

Like adding extra DNA, throwing extra pipes at a plumbing leak is a great way to make a bad problem worse.

But for the cell, the results are not as hilarious as they were for Moe, Larry, and Curly. That extra DNA can lead to deadly genetic diseases.

A yeast cell needs Isw1p to keep the cell from bringing in the Three Stooges to mess up its DNA and potentially cause devastating genetic diseases. If it turns out the same is true in people, then once again yeast will have shown us how human genetic diseases might happen. And perhaps provide a target for us to go after to prevent these diseases from happening. #APOYG!

by Barry Starr, Ph.D., Barbara Dunn, Ph.D., and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: chromatin remodeling, DNA repair, ISW1, nucleosome, RNA polymerase II

Apply Now for the 2018 Yeast Genetics and Genomics Course

March 16, 2018

For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists. The application deadline is April 15th, so don’t miss your chance! Find all the details and application form here. This year’s instructors – Grant Brown, Greg Lang, and Elçin Ünal – have designed a course (July 24 – August 13) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:

• Finding and Analyzing Yeast Information Using SGD
• Isolation and Characterization of Mutants
• Yeast Transformation, Gene Replacement by PCR, and Construction and Analysis of Gene Fusions
• Tetrad Analysis
• Genome-scale Screens Using Synthetic Genetic Array (SGA) Methodology    
• Deep Sequencing Applied to SNP Mapping and Deep Mutational Scanning
• Exploring Synthetic Biology with CRISPR/Cas9-directed Engineering of Biosynthetic Pathways
• Computational Methods for Data Analysis
• Modern Cytological Approaches Including Epitope Tagging and Imaging Yeast Cells Using Fluorescence Microscopy

Techniques have been summarized in a completely updated course manual, which was recently published by CSHL Press.

Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.

Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!

The Yeast Genetics & Genomics Course is loads of fun – don’t miss out!

 

Categories: Announcements

Don’t Ignore the Scullery Maid, Hobbits, or Pol III

February 13, 2018

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In the Disney story, Cinderella is ignored as the scullery maid. Most of the attention falls on her step-sisters.

This is, of course, a mistake. The best person by far is Cinderella and she should not be ignored because of her position in life.

The RNA polymerases in a eukaryotic cell are similar except that one is the star and two are the scullery maids. RNA polymerase II (Pol II), the reader of protein encoding genes, gets most of the attention. Pol I and Pol III, the enzymes that make RNAs that aren’t usually translated into proteins, are mostly relegated to supporting roles.

In a study in Nature, Filer and coworkers show that ignoring Pol III can be as big a mistake as ignoring Cinderella. If you want to live a longer life that is.

Just as there were hints of Cinderella’s specialness, so too there were hints that perhaps Pol III plays an important role in longevity. That hint goes by the name of TORC1.

Previous work had shown that inhibiting TORC1 can increase the lifespan of beasts ranging from yeast to mice. Given that TORC1 is a strong activator of Pol III, Filer and coworkers set out to determine if inhibiting Pol III could do the same thing.

As any good researcher should, they started out by looking in our favorite model organism, the yeast Saccharomyces cerevisiae.

Pol III is pretty complicated—it is made up of 17 different subunits encoded by 17 different essential genes. These authors went after the largest subunit, C160, encoded by the RPO31 (RPC160) gene.

To control its level of expression, they fused an auxin-inducible degron to the protein. This fusion protein is degraded in the presence of indole-3-acetic acid (IAA) and E3 ubiquitin ligase from the rice Oryza sativa.

This means that there should be less Pol III in a cell when IAA is around. Western blot analysis confirmed that this was indeed the case.

The authors found that yeast with less Pol III had a longer chronological lifespan. In other words, each yeast cell survived longer. Having less Pol III did not significantly increase replicative lifespan though; they did not bud more often over their lifetime.

These authors did not get as significant a result when they targeted the largest subunit of Pol II, RPO21 (RPB220). The strain appeared to do better in the presence of IAA compared to the control strain, suggesting that targeting Pol II had a small effect on longevity; but it was nothing like was seen with Pol III.

They next turned to a couple of model organisms with more cells—nematodes, also known as C. elegans, and fruit flies, or Drosophila.

When Filer and coworkers used RNA interference (RNAi) to knock down the amount of the largest subunit of Pol III, rpc-1, in C. elegans, the worm lived longer. And when they generated a strain of Drosophila which had only one working copy of dC53 (CG5147), a gene that encodes a Pol III subunit, these fruit flies lived longer too. Lowering the amount of Pol III is a good thing if you want to live longer!

Previous work had shown that the gut often controls longevity in C. elegans. These authors showed that the same holds true for Pol III reduction. Targeting just the Pol III in the gut was sufficient to give this little worm a longer life.

The same turned out to be true in the fruit fly. Using RNAi against two different Pol III subunits in the Drosophila gut was enough to have extend these flies’ lives. Targeting Pol III in the fat body did not have the same effect, and targeting Pol III in neuronal cells extended life only a little.

So reducing Pol III in the gut is good enough for flies and worms to live longer. In fact, reducing Pol III activity had as much effect on longevity as does inhibiting TORC1. Scientists ignore this hidden gem at their own risk!

Since Pol III is ignored, but dangerous for lifespan, perhaps a better analogy than Cinderella might be Sauron and the hobbits of the Shire from the Lord of the Rings Trilogy. Sauron ignores the hobbits who hold the key (or the ring) to Sauron’s shortened lifespan.

If Sauron had paid attention, he’d still be lording it over Middle Earth at the end of the third book. Just as we might live longer if we start paying more attention to that hidden danger—excessive Pol III activity, possibly in the gut.

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

Categories: Research Spotlight

Tags: Longevity, RNA polymerase III, TORC1

Strung Out Chromosomes and Coyotes

January 29, 2018

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As Wile E. Coyote knows, when setting a bear trap, you need to make sure the tension is just right. As you can see in this video, if there isn’t enough, then the Road Runner gets away:

And of course, Wile E. ends up getting caught in the trap!

Like our super-genius coyote friend, Saccharomyces cerevisiae cells need to make sure there is the right amount of tension sometimes, too. One of those times, for example, is tension on their chromosomes at the metaphase stage of cell division.

If the chromosomes aren’t attached to the spindle in just the right way, the cell will not let mitosis move from metaphase to anaphase. The cell won’t put down the birdseed until it “knows” the trap is set properly.

While Wile E. Coyote probes the tension by eyeballing it, yeast does it in a much more thorough way. Two key pieces of the sensing machinery in yeast are the tension sensing motif (TSM) from histone H3, ⁴²KPGT, and the Shugoshin 1 protein, Sgo1p.

The current model is that the TSM of histone H3 acts as an anchoring point or a landing pad for Sgo1p, the protein responsible for sensing that there is the right amount of tension between chromosomes and the spindle/kinetochore. When the TSM is mutated with, for example, a G44S allele, the idea is that Sgo1p can no longer bind causing chromosomes to sometimes segregate incorrectly.

Previous work had shown that mutating Gcn5p can partially rescue the effects of the G44S allele. In a new study in GENETICS, Buehl and coworkers provide evidence that the reason it can has to do with the tail of histone H3. Basically, this tail can act as a secondary anchor if the TSM, the first anchor point, is mutated. But the tail can only work if it is not acetylated by Gcn5p.

It is as if Wile E. Coyote’s bear trap is broken so he has to fashion a new trigger to get it to snap. When the TSM of H3 is gone, yeast can fashion a new sensor by eliminating the acetylation on the tail of the H3 histone.

Buehl and coworkers focused on the tail because it is a known target of Gcn5p acetylation. In particular, there are four lysine residues in the tail—K9, K14, K18, and K23—that are acetylated by Gcn5p to varying degrees. The most acetylated is K14 followed by K23 and K18. K9 is hardly acetylated at all.

When they deleted the histone tail, they found that the effects of the TSM mutation, G44S, were more severe. But when they mutated the four lysines all at once, the effects of G44S weren’t as bad. This is consistent with the unacetylated tail serving as a landing pad for Sgo1p in the absence of the TSM. No tail, no binding, leading to chromosome mis-segregation.

As might be expected if this was linked to Gcn5p, each individual lysine mutant had effects that corresponded to their level of acetylation by Gcn5p. So K14A did the best job of partially rescuing the G44S allele, K18A and K23A did an okay job and K9A had very little effect.

Buehl and coworkers provide three additional sets of experiments to show that G44S causes chromosome mis-segregation and that K14A can partially rescue it. There is only time to discuss one of these, but you can read the paper for the other two.

This strategy uses the idea that diploids cannot mate because of a repression function on each copy of chromosome III (chrIII). If a diploid loses one of its chrIII’s, then it will be able to mate.

As expected, the authors found that both wild type and the K14A mutant had very few diploids that could mate. But it was a different story for the G44S mutant—it had many more diploids able to mate, presumably due to the loss of one chrIII. The double mutant K14A/G44S had more diploids mate than wild type but less that G44S on its own.

This is what we would expect if K14A can only affect tension sensing in a TSM mutant. It would do little on its own but could partially rescue the TSM mutant G44S.

The authors next used chromatin immunoprecipitation (ChIP) assays to directly show that the K14A mutation in histone H3 allowed Sgo1p to bind when the TSM was mutated. There was one hitch though—Sgo1p bound most everywhere and not just at the pericentromeres where it usually does.

In a final set of experiments, the authors used GST-pulldowns to show a direct interaction between the H3 tail and Sgo1p. To their surprise, they found that acetylating the tail did not affect Sgo1p binding. They suggest that perhaps the acetylated tail interacts with something that blocks Sgo1p from binding.

Yeast has a backup just in case its main way of sensing tension between the spindle/kinetochore and chromosome, the TSM of histone H3, breaks down. In that case, it can use the tail of histone H3, assuming it is not acetylated.

If only Wile E. Coyote included backups like this in his crazy plans! Then he could finally dine on that tasty Road Runner he is always chasing.

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

Categories: Research Spotlight

Tags: acetylation, chromosome segregation, Histone H3, metaphase

Exploding Yeast to Protect the Wild

January 22, 2018

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In the first Kingsman movie, Samuel L. Jackson plants little bombs into the necks of people who will follow him to his “ark”. If they disobey him, Jackson can simply blow up their heads.

In an amazing scene from the movie, our heroes get a hold of the “detonator” and blow up the heads of every one of Jackson’s followers. As you can see below, the director uses cool colors to make it more fabulous than gruesome:

In a new study out in Nature Communications, Maselko and coworkers explode misbehaving yeast instead of people’s heads. (Click here for an incredibly cool video of it happening…so cool!)

These researchers have created a strain of yeast that can only form diploids within the same strain. If instead it mates and forms a diploid with a yeast outside of its strain, the resulting diploids explode.

These authors aren’t being strainist—they are creating a proof of principle organism that could potentially solve the problem of gene transfer from genetically modified organisms (GMOs) to wild organisms.

Assuming a similar idea works in plants, then the idea is that a GMO like an herbicide-resistant GM plant could not pass its herbicide-resistance to its wild cousin. The resulting plants would explode (or perhaps die in a bit less spectacular fashion). The herbicide-resistant gene would not escape into the wild population because none of the resulting plants could survive.

And that isn’t this approach’s only possible uses. It might be used to kill off disease carrying mosquitoes or controlling invasive species. Watch out sea lamprey!

The basic idea is that the engineered species makes a “poison” that it is immune to. If it mates with a wild species, it passes that poison on to the resulting “children.” These progeny are not immune and so die.

The poison in this case is a CRISPR/Cas9-inspired transcription activator. This activator turns a gene up so much that it kills the yeast. But the catch is that the activator’s binding site is only found in the wild type genome and not the engineered genome.

So the engineered strain carries around this deadly poison that does not affect it. But when this yeast mates with a strain that has the binding site, the resulting diploid yeast has the activator binding site, meaning that the silent killer can now bind and kill the diploid.

For their proof of principle experiments, Maselko and coworkers used a well-tested and available CRISPR/Cas9 transcriptional activator that involves dCas9-VP64 and a single guide RNA aptamer binding MS2-VP64. Previous work has shown that this is a very powerful activator.

Next they turned to the Saccharomyces Genome Database (SGD) to find genes that have been shown to be lethal when overexpressed. They created at least one guide RNA for each of the 8 genes and found that one of the guide RNAs that targeted the ACT1 promoter was lethal. The yeast made actin until it exploded (think the man in Monty Python’s Meaning of Life who eats so much he explodes).

Now they were ready to create their engineered strain. They used CRISPR/Cas9 to engineer a single nucleotide change such that their activator could no longer bind near the ACT1 gene. This strain survived when the activator was introduced.

But when they mated this strain with a wild type strain, they got almost no survivors. The resulting diploids grew fat on overproduced actin and exploded!

Note that there were a few survivors…as Jeff Goldblum famously said in Jurassic Park, “Life will find a way.” The survivors appeared at a frequency of 4.83 × 10⁻³ with the most common reason found being a mutation in the activator binding site.

This survival rate is one area of obvious concern. Given the genetic variability of wild populations, there may be a few individuals that happen to have a mutation that is resistant to the activator. These immune individuals would then go on to found a new population.

The authors suggest that targeting a species that has gone through a recent bottleneck to decrease genetic variability might help. Another possibility might be to target multiple genes making the likelihood of an individual having and/or developing multiple mutations exceedingly unlikely. (This is the strategy used to target HIV, a virus with a high mutation rate.)

These authors have created an elegant system reminiscent of the one created by Baron Harkonnen in the novel Dune. In the book, a character named Thufir Hawat is given a slow acting poison by the Baron. Hawat can be controlled because he needs the antidote each day to survive. Once the antidote is removed, he dies.

Here the researchers have given the poison and engineered the antidote into their new strain. When this strain mates with a strain lacking the antidote, like a wild strain, most of the progeny die. Like Hawat, the yeast die for the good of mankind. And hopefully those pesky weeds can be prevented from picking up transgenes too.

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

Categories: Research Spotlight

Tags: CRISPR, horizontal gene transfer, synthetic biology

Moonlighting Proteins Do Double Duty

January 11, 2018

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While most people only know her as the friendly waitress at the local diner, it is by night that Louise gets to live her passion. At night, she is a mime.

If she loses her voice, Louise will struggle as a waitress but do fine as a mime. It is a different story when she gets walloped with a nasty flu and can’t get out of bed. Now she can’t wait tables by day or be stuck in an imaginary box by night.

In a new study out in GENETICS, Espinosa-Cantu and coworkers get to explore which enzymes in our devoted pal Saccharomyces cerevisiae are like Louise in that they do two unrelated jobs. They set out to systematically identify enzymes that have both a catalytic job and a job unrelated to its catalytic function. In other words, they set out to find enzymes that moonlight at second jobs.

Other researchers have stumbled upon these moonlighting enzymes in the course of their regular studies. Something along the lines of stumbling upon Louise struggling against an imaginary wind at a local park.

The difference here is that Espinosa-Cantu and coworkers systematically looked for dual-function enzymes. They did this by comparing the effects of knocking out the entire protein vs. just knocking out the catalytic activity of the enzyme with a single point mutation. They compared Louise having laryngitis vs. her being bedridden with illness.

They found that 4 out of the 11 enzymes they looked at did indeed have additional jobs unrelated to their catalytic function. These enzymes were both mimes and waitresses.

They started out looking at enzymes involved in amino acid biosynthesis mostly because it is easy to test them for loss of function. The yeast can’t grow in the absence of the amino acid the enzyme helps synthesize!

Next, like every good yeast researcher should, they turned to the Saccharomyces Genome Database (SGD) to identify 86 genes that when knocked out, caused the mutant yeast strain to not grow in the absence of an amino acid. They winnowed this list to 18 by focusing only on those enzymes with well-characterized catalytic sites and that were annotated with a single molecular function.

Then, they needed to have versions of these enzymes that were essentially identical to wild type except that they could not catalyze their reaction. They focused on single amino acid mutants that wiped out catalytic activity. The authors got this to work in 15 of their candidate enzymes.

They were now ready for the big experiment. They started out with a strain deleted for one of the enzymes and then added back either a wild type version or a catalytically inactive mutant version on a low copy (CEN-ARS) plasmid. They also included a third strain that had just an empty plasmid (their knockout strain).

They compared the growth of these three strains under 19 different growth conditions and looked for conditions where the knockout strain had a larger effect on growth compared to the strain with the catalytic mutation. The idea here is that if an enzyme does more than one job, knocking it out should have a larger effect than just disabling its enzymatic activity.

Before analyzing their results, the authors wanted to make sure that their wild type strain, the knockout strain with the deleted gene added on a CEN-ARS plasmid, grew equivalently to a true wild type strain. This was only true in 11/15 of the strains and so their analysis focuses on these 11 enzymes.

Espinosa-Cantu and coworkers found no difference in growth rate with all three strains for 38.6% of the growth rate comparisons. Under these conditions, the enzyme isn’t doing much for the growth rate of the cells.

In 233 growth conditions, the enzyme did matter for growth and in 70% of these, there was no difference between the knockout and catalytically inactive strains. Presumably under these conditions the enzymes have a single job—their catalytic function.

But this left 30% of cases where there was a difference suggesting that under these conditions, the enzymes have more than one job. Most of these involved 4 of the 11 genes—ALT1, BAT2, ILV1, and ILV2.

They decided to focus in more detail on one of these, ILV1. To figure out what else Ilv1p might be doing besides its threonine deaminase job, they generated two separate strains of double knockouts with 3,878 non-essential genes. The first set of 3,878 strains had a deletion of ILV1 while the second set had a catalytically inactive form of ILV1.

When they looked at the cases where the ILV1 knock out strain was more severely affected compared to the catalytically inactive strain, they found that 187 out of 345 negative interactions had at least some non-catalytic component.  The non-catalytic interactions were enriched for gene regulation with chromatin modification being one of the most striking.

These interactions put the non-catalytic role of ILV1 in a category more similar to stress response, protein sorting and chromatin remodeling genes than to other metabolic genes. This suggests that Ilv1p is both involved in amino acid metabolism and stress response. Quite a different moonlighting gig compared to its day job!

So researchers need to be careful when drawing conclusions from their knockout experiments. Sometimes the effect you are seeing is unrelated to the function you are studying. Sometimes when a waitress falls ill, the mime performance is affected as well.

No this isn’t a show about ILV1.

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

Categories: Research Spotlight

Tags: pleiotropy, protein moonlighting, systems genetics

Happy Holidays from SGD!

December 22, 2017

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We want to take this opportunity to wish you and your family, friends and lab mates the best during the upcoming holidays.

Stanford University will be closed for two weeks from December 23rd through January 7th. Regular operations will resume on Monday, January 8, 2018.

Although SGD staff members will be taking time off, please rest assured that the website will remain up and running throughout the winter break, and we will attempt to keep connected via email should you have any questions.

Happy Holidays and best wishes for all good things in the coming New Year!

Categories: Announcements

SGD December 2017 Newsletter

December 21, 2017

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SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This December 2017 newsletter is also available on the community wiki.

Categories: Newsletter

Retooling Yeast Factories

December 13, 2017

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Imagine you own a rifle factory and you want to retool it to make solar panels instead. Or vice versa.

It wouldn’t make a lot of sense to make small, incremental changes. Make one change, test to see how it works, make a second change, test to see how that works, and so on.

Not only is this inefficient and time consuming, but you may not end up with the best factory for making your new product. The best approach is to rip out the old machinery and replace it all at once. Or at the very least make many changes at a time.

The same sorts of problems exist for cellular factories—microbes we engineer to churn out useful products. Ideally we would want to get to an optimized, retooled cellular factory as soon as possible by making as many changes as we can at once. And yet, that is not how we currently do it.

Most of the time when we repurpose a microbial organism like Saccharomyces cerevisiae to make something new, we do it the slow way. We either do a screen to find genes that affect the process or change a known gene and analyze the results. We then use this mutant strain as a starting point to repeat the process. Over many cycles, we hopefully get to a yeast strain that can make something new or more efficiently.

Not only is this time consuming and inefficient, but we might miss some synergies that can happen when two or three genes are tweaked at once. This is no way to retool a factory!

In a new study out in Nature Communications, Lian and coworkers came up with a clever way to make many changes all at once in yeast. And they aren’t limited to just deleting genes either. They can transcriptionally activate, transcriptionally interfere, or delete specific genes. Which is where they got their clever acronym for their process—CRISPR-AID.

As you can tell from the name, their system uses the seemingly ubiquitous gene-editing tool CRISPR. And they use it in some very cool ways to get all three processes happening in a cell at the same time with different genes.

Conventional CRISPR works through a nuclease being guided by an aptly named guide RNA (gRNA) to a precise place in cell’s DNA through base pairing between the DNA and the gRNA.  Once there, the nuclease makes a double-stranded break and at least in yeast, the cell invariably repairs it using homologous directed repair (HDR). (Other eukaryotes tend to “fix” their DNA with the more error prone non-homologous end joining [NHEJ] pathway. Yes, yeast is superior in yet another way!)

Scientists have been able to tweak this system to activate or repress gene transcription by knocking out the nuclease activity of the CRISPR protein and adding back either an activation or repression domain. Now they have an RNA-guided, sequence specific transcription activator or repressor.

This is a great tool chest of gene editing tools but there is one problem—how to get the right protein to the right spot when all three use a gRNA for specificity. Like Mork from Ork turning to Mindy to help guide him on Earth, Lian and coworkers turned to a PAM to help guide these proteins to the right place in the yeast genome.

The protospacer adjacent motif (PAM) is a small part of the gRNA that binds the DNA and helps to pry it open so the rest of the gRNA can bind. Every gRNA has to have a PAM or the CRISPR protein won’t bind.

It turns out that different bacteria have different CRISPR systems that use different PAMs. The idea then is to use a CRISPR protein from different bacteria for each of the three different functions of the CRISPR-AID system.

The most common form of CRISPR protein is Cas9 from Streptococcus pyogenes (Sp), which has NGG for its PAM sequence. Lian and coworkers used a nuclease deficient form of this protein attached to a repressor domain to create their transcription interference tool dSpCas9-RD1152. (The d means the nuclease in inactivated, the Sp is the bacteria it came from, and the RD1152 is the repression domain.)

For their deletion tool they turned to the Staphylococcus aureus (Sa) Cas9, SaCas9, which has NGRRT or NGRRN for its PAM sequence.

They turned to a different CRISPR protein, Cfp1 from Lachnospiraceae bacterium ND2006 (Lb), for their activation tool. This protein recognizes TTN as its PAM. They attached an activation domain to a nuclease-deficient form to generate dLbCfp1-VP.

All three proteins were stably integrated into a yeast cell. Now that their toolbox was full and their yeast cell ready, Lian and coworkers set out to show that these tools were effective.

They first used it on a known system, making beta-carotene in yeast. Previous work had shown that increasing the expression of the HMG1 gene, decreasing the activity of ERG9, and deleting ROX1 led to increased beta-carotene production.

They used CRISPR-AID to accomplish all three in one fell swoop. Not only did expression from HMG1 go up, expression from ERG9 go down, and ROX1 get deleted, but the resulting yeast also made more beta-carotene. They got a 2.8-fold increase in beta-carotene compared to only a 1.7 fold increase by hitting just one of the genes.

They next set out to increase expression of recombinant Trichoderma reesei endoglucanase II (EGII). Previous work had identified 14 genes that increased expression when activated, 17 that increased expression when inhibited and 5 that increased expression when deleted. Each of these was done as a single gene experiment.

Lian and coworkers created a library of gRNAs that could target each of the 1620 possible combinations to see if any combination would lead to a synergistic increase in activity. They found a combination that did just that—increased expression of PDI1, decreased expression of MNN9, and deletion of PMR1 led to the highest yield of EGII. The increased expression of EGII was beyond what any of the three did on their own.

So the system appears to be working well. Multiple targets can be upregulated, downregulated or deleted all at once.

While we are getting closer to being able to retool microbial factories as efficiently as brick and mortar ones, we aren’t there yet. That will require a deeper understanding of how a particular microbial organism works. And there is no better understood eukaryote than our old friend S. cerevisiae.

Like a good PAM sequence, Mindy helps Mork find his place in the world.

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

Categories: Research Spotlight

Tags: CRISPR, gene editing, microbial factory

A SRTain Surprise in a Lipid Droplet

December 04, 2017

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Barney Stinson from the sitcom “How I Met Your Mother” is right—the accidental curly fry in your plate of fries is a wonderful thing. It is unexpected but adds immensely to the French fry eating experience.

In a new study in GENETICS, Hoffmann and coworkers show that spore wall lipid droplets, organelles used to move neutral lipids to the cell wall in the yeast Saccharomyces cerevisiae, have a surprise component, too. Instead of an errant curly fry in an order of fries, the greasy droplet has long chain polyisoprenoids made by Srt1p and its partner, Nus1p.

And in an unexpected twist, they are not used to glycosylate a protein as most polyprenols do. No, they upregulate the activity of a key protein involved in spore wall synthesis—Chs3p. Polyprenols may have more varied roles than scientists thought. A pleasant surprise indeed.

When a diploid yeast cell hits hard times, it undergoes sporulation to make sturdy spores to ride them out. And the spores’ sturdiness comes from the spore cell walls.

Yeast need many factors to make these resistant walls. One is Chs3p, the chitin synthase that makes the chitin that is used to make one of the four spore cell wall layers. Another is the lipid droplet that brings a variety of lipids and proteins to the site of the expanding cell wall.

Hoffmann and coworkers used transmission electron microscopy (TEM) to show that spores from strains deleted for SRT1 lack the chitosan layer of their cell walls. Additional experiments showed that this strain is missing this layer because it fails to make chitin as opposed to failing to be able to assemble chitin properly.

The Srt1p/Nus1p complex is a cis–prenyltransferase—it adds 5-carbon isopentyl groups to farnesyl diphosphate to make polyprenols. In the next set of experiments, these researchers showed that an enzymatically inactive mutant of Srt1p, Srt1p-D75A, did not make the chitosan layer as well. This suggested that it is the long chain polyisoprenoids that Srt1p/Nus1p synthesizes that matters for spore wall formation.

In the final set of experiments, Hoffmann and coworkers showed that extracts lacking Srt1p had decreased chitin synthase activity. In other words, they showed that the activity of Chs3p is dependent on Srt1p.

Taken together, these results suggest that polyprenols like long chain polyisoprenoids may do more than simply facilitate the glycosylation of proteins. Here, they upregulate the activity of a protein involved in cell wall synthesis.

And we aren’t just talking yeast either. Most every living thing, including people, have polyprenol-derived dolichols which carry sugars to facilitate protein glycosylation. It may be that these types of molecules also have hidden, unexpected roles. Researchers may want to give these molecules a second look to see if they missed that extra curly fry.

Taking a man’s bonus curly is inexcusable.

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

Categories: Research Spotlight

Tags: cell wall, chitin, chitosan, spore, SRT1

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