CRISPR/Cas9: More Than a Genome Editor

By Rebecca Delker, PhD

 

The bacterial defense system, CRISPR/Cas9, made huge waves in the biomedical community when the seemingly simple protein-RNA complex of Type II CRISPR systems was engineered to target DNA in vitro and in complex eukaryotic genomes. The introduction of double-strand breaks using CRISPR/Cas9 in a targeted fashion opened the portal to highly affordable and efficient site-specific genomic editing in cells derived from yeast to man.

 

To get a sense of the impact CRISPR technology has had on biological research, one simply needs to run a search of the number of publications containing CRISPR in the title or abstract over the past handful of years; the results practically scream in your face. From 2012, the year of the proof-of-principle experiment demonstrating the utility of engineered Cas9, to 2015, CRISPR publications rose steadily from a mere 138 (in 2012) to >1000 (at the time of this post). Publications more than doubled between the years of 2012 and 2013, as well as between 2013 and 2014. Prior to the use of CRISPR as a technology, when researchers studied the system for the (very cool) role it plays in bacterial defense, publications-per-year consistently fell below 100. In other words, it’s a big deal.

 

In fact, during my 10 years at the bench I have never witnessed a discovery as transformative as CRISPR/Cas9. Overnight, reverse genetics on organisms whose genomes were not amenable to classical editing techniques became possible. And with the increasing affordability of high-throughput sequencing, manipulation of the genomes of non-model organisms is now feasible. Of course there are imperfections with the technology that require greater understanding to circumvent (specificity, e.g.), but the development of CRISPR as a tool for genomic engineering jolted biological research, fostering advances more accurately measured in leaps rather than steps. These leaps – and those expected to occur in the future – landed the discoverers of CRISPR/Cas9 at the top of the list of predicted recipients of the Nobel Prize in Chemistry; though they didn’t win this year (the award went to researchers of the not-totally-unrelated field of DNA repair), I anticipate that a win lies ahead. The rapid success of CRISPR genome editing has also sparked patent battles and incited public debate over the ethics of applying the technology to human genomes. With all of the media attention, it’s hard not to know about CRISPR.

 

The transformative nature of CRISPR/Cas9 does not, however, end with genome editing; in fact, an even larger realm of innovation appears when you kill the enzymatic activity of Cas9. No longer able to cut DNA, dead Cas9 (dCas9) becomes an incredibly good DNA-binding protein guided to its target by a programmable RNA molecule (guide RNA, gRNA). If we think of active Cas9 as a way to better understand genes (through deletions and mutations), then dCas9 is the route to get to know the genome a bit better – a particularly enticing mission for those, including myself, invested in the field of Genomics. From high-throughput targeted gene activation and repression screens to epigenome editing, dCas9 is helping scientists probe the genome in ways that weren’t possible before. Here, I put forth some of the best (in my humble opinion) applications, actual and potential, of CRISPR technology that go beyond genome editing.

 

Cas9 and Functional (Epi)Genomics

 

For many years the genome was considered as the totality of all genes in a cell; the additional junk DNA found was merely filler between the necessary gene units, stitching together chromosomes. We’ve come a long way since this naiveté, especially in recent years. We understand that the so-called junk DNA contains necessary regulatory information to get the timing and position of gene expression correct; and now, more than ever, we have a greater appreciation for the genome as a complex macromolecule in its own right, participating in gene regulation rather than acting as a passive reservoir of genetic material. The genome, it has been shown, is much more than just its sequence.

 

The epigenome, consisting of a slew of modifications to the DNA and the histones around which the DNA is wrapped, as well as the 3D organization of the genome in the nucleus, collaborates with DNA binding proteins to accurately interpret sequence information to form a healthy, functional cell. While mutations and/or deletions can be made – more easily, now, with Cas9 – to genomic sequences to test functionality, it is much harder to conduct comparable experiments on the epigenome, especially in a targeted manner. Because of the inability to easily perturb features of the epigenome and observe the consequences, our understanding of it is limited to correlative associations. Distinct histone modifications are associated with active versus inactive genes, for example; but, how these modifications affect or are affected by gene expression changes remains unknown.

 

Taking advantage of the tight binding properties of dCas9, researchers have begun to use the CRISPR protein as a platform to recruit a variety of functionalities to a genomic region of interest. Thus far, this logic has most commonly been employed to activate and/or repress gene expression through recruitment of dCas9 fused to known transcriptional activator or repressor proteins. Using this technique, scientists have conducted high-throughput screens to study the role of individual – or groups of – genes in specific cellular phenotypes by manipulating the endogenous gene locus. And, through a clever extension of the gRNA to include a hairpin bound by known RNA-binding proteins, the targeted functionality has been successfully transferred from dCas9 to the gRNA, allowing for simultaneous activation and repression of independent genes in the same cell with a single dCas9 master regulator – the beginnings of a simple, yet powerful, synthetic gene circuit.

 

Though powerful in its ability to decipher gene networks, dCas9-based activation and repression screens are still gene-centric; can this recruitment technique help us better understand the epigenome? The first attempts at addressing this question used dCas9 to target histone acetyltransferase, p300, to catalyze the acetylation of lysine 27 on histone 3 (H3K27) at specific loci. The presence of H3K27 at gene regulatory regions has been known to be strongly associated with active gene expression at the corresponding gene(s), but the direction of the histone modification-gene expression relationship remained in question. Here, Hilton et al. demonstrate that acetylation of regulatory regions distal to gene promoters strongly activates gene expression, demonstrating causality of the modification.

 

More recently, recruitment of a dCas9-KRAB repressor fusion to known regulatory regions catalyzed trimethylation of lysine 9 on histone 3 (H3K9) at the enhancer and associated promoters, effectively silencing enhancer activity. Though there have only been a few examples published, it will likely not be long until researchers employ this technique for the targeted analysis of additional epigenome modifiers. Already, targeted methylation, demethylation and genomic looping have been accomplished using the DNA-binders, Zinc Finger Nucleases and TALEs. With the increased simplicity in design of gRNAs, dCas9 is predicted to surpass these other proteins in its utility to link epigenome modifications with gene expression data.

 

Visualization of Genomic Loci

 

When you treat dCas9 as a bridge between DNA and an accessory protein, just as in the recruitment of activators, repressors and epigenome modifiers, there are few limits to what can be targeted to the genome. Drawing inspiration from the art of observation that serves as the foundation of scientific pursuit, researchers have begun to test whether dCas9 can be used to visualize genomic loci and observe their position, movements, and interactions simply by recruiting a fluorescent molecule to the locus of interest.

 

This idea, of course, is not entirely new. In situ hybridization techniques (ISH, and its fluorescent counterpart, FISH) have been successfully used to label locus position in fixed cells but cannot offer any information about the movement of chromosomes in living cells. Initial studies to conquer this much harder feat made use of long tracts of repetitive DNA sequence bound by its protein binding partner fused to fluorescing GFP; though surely an advance, this technique is limited because of the requirement to engineer the repetitive DNA motifs prior to imaging.

 

To circumvent this need, researchers have recently made use of TALEs and dCas9 (and here) carrying fluorescent tags to image unperturbed genomic loci in a variety of live cell cultures. The catch is that both TALEs and dCas9 perform much better when targeting repetitive regions, such that multiple copies of the fluorescent molecule are recruited, enhancing the intensity of the signal. Tiling of fluorescent dCas9 across a non-repetitive region using 30-70 neighboring gRNAs (a task made much more feasible with CRISPR versus TALEs) can similarly pinpoint targeted loci, albeit with much higher background. As is, the technique lacks the resolution desired for live imaging, but current advances in super-resolution microscopy and single-molecule tracking, as well as improvements in the brightness of fluorescent molecules available, will likely spur improvements in dCas9 imaging in the coming years.

 

Finally, dCas9 is not only useful in live cells. CASFISH, an updated Cas9-mediated FISH protocol, has been successfully used to label genomic loci in fixed cells and tissue. This updated version holds many benefits over traditional FISH including a streamlined protocol; but, most notably, CASFISH does not require the denaturation of genomic DNA, a necessary step for the hybridization of FISH probes, eliminating positional artifacts due to harsh treatment of the cells. Unfortunately, as of now, CASFISH also suffers from a need for repetitive sequences or tiling of gRNAs to increase signal intensity at the locus of interest.

 

Targeting RNA with Cas9

 

From cutting to tagging to modifying, it is clear that Cas9 has superstar potential when teamed up with double-stranded DNA (dsDNA); however, recent data suggests that this potential may not be limited to DNA. Mitchell O’Connell and colleagues at Berkeley found that Cas9 could bind and cleave single-stranded RNA (ssRNA) when annealed to a short DNA oligonucleotide containing the necessary NGG sequence. In addition, the authors made use of dCas9 and biotin-tagged gRNA to capture and immobilize targeted messenger RNA from cell extract. Though it remains to be shown, this proof-of-principle binding of dCas9 suggests that it is plausible to recruit a variety of functionalities to RNA as has been done for dsDNA. Recruitment of RNA processing factors through Cas9 could potentially enhance translation, generate known RNA editing events (deamination, e.g.), regulate alternative splicing events, or even allow visualization of RNA localization with conjugated fluorescent molecules. Again, each of these processes requires no modification to the RNA sequence or fixation, both of which can disrupt normal cell physiology.

 

Improving CRISPR Technology

 

The development of CRISPR technology, particularly the applications discussed here, is still in its infancy. It will likely take years of research for Cas9 and dCas9 to reach their full potential, but advances are underway. These developments pertain not only to the applications discussed here, but also genome engineering.

 

Specificity of Cas9

 

Cas9’s biggest flaw is its inability to stay focused. Off-target (OT) binding (and here) of Cas9 and DNA cutting have been reported and both present problems. With particular relevance to dCas9-based applications, promiscuous binding of Cas9 to regions of the genome that contain substantial mismatches to the gRNA sequence raises concerns of non-specific activity of the targeted functionality. Efforts to reduce OT binding are needed to alleviate these concerns, but progress has been made with the finding that truncated gRNA sequences are less tolerant of mismatches, reducing off-target Cas9 activity, if not also binding.

 

Temporal Precision of Cas9

 

One of the most exciting developments in dCas9 genome targeting is the potential to manipulate the genome and epigenome in select cell populations within a whole animal to gain spatial resolution in our understanding of genome regulation; however, as we have learned over the years, gene expression patterns don’t only change with space, but also time. A single cell, for example, will alter its transcriptome at different points during development or in response to external stimulus. The development of split versions of Cas9 (and dCas9), which require two-halves of the protein to be expressed simultaneously for function, will not only improve spatial specificity of Cas9 activity but holds the potential to restrict its activity temporally. Drug-inducible and photoactivatable (!) versions of split Cas9 restrict function to time windows of drug treatment or light activation, respectively. In addition, a ligand-sensitive intein has been shown to temporally control Cas9 activity by releasing functional Cas9 through protein splicing only in the presence of ligand.

 

Expanding the CRISPR Protein Repertoire

 

Finally, CRISPR technology will likely benefit from taking all of the weight off of the shoulders of Cas9. Progress toward designing Cas9 molecules with altered PAM specificity, as well as the isolation of Cas9 from different species of bacteria, has helped expand the collection of genomic sites that can be targeted. It has also enabled multiplexing of orthogonal CRISPR proteins in a single cell to effect multiple functions simultaneously. More recently, the Zhang lab isolated an alternative type II CRISPR protein, Cpf1, purified from Francisella novicida. Cas9’s new BFF is also able to cut genomic DNA (as shown in human cells), but in a slightly different fashion than Cas9, generating sticky overhangs rather than blunt ends. Cpf1 also naturally harbors an alternate PAM specificity; rather than targeting sequences upstream of NGG, it prefers T-rich signatures (TTN), further expanding the genomes and genomic sites that can be targeted.

 

CRISPR/Cas9 has already proven to be one of the most versatile tools in the biologist’s toolbox to manipulate the genomes of a variety of species, but its utility continues to grow beyond these applications. Targeting Cas9 to the mitochondria rather than the nucleus can specifically edit the mitochondrial genome, with implications for disease treatment. Cas9 has been used for in vitro cloning experiments when traditional restriction enzymes just won’t do. And, by directly borrowing the concept of Cas9 immunity from bacteria, researchers have enabled enhanced resistance to viruses in plants engineered with Cas9 and gRNAs. While we ponder what innovative technique will come next, it’s important to think about how this cutting-edge technology that promises to bolster both basic and clinical research came to be: this particular avenue of research was paved entirely by machinery provided by the not-so-lowly bacteria. That’s pretty amazing, if you ask me.

Drosophila Diaries: Ken and Barbie

By Michael Burel

 

The holidays are upon us, people. This is not a drill. While the greatest gift of all during the holiday season is giving rather than receiving, you can’t help but remember how amazing (and admittedly materialistic) it is to receive things from others. “Things, for free?!” Yes, things! For free! Such a life exists even for graduate students who exploit this chance to forgo extravagance in exchange for desperately needed life necessities: Tupperware, socks, food, shelter, social interaction, separation anxiety from work, relief from the dark ends of the seemingly infinite thesis tunnel. You know, the basics.

 

Perhaps one of the most interesting side effects of the holiday season is nostalgia, that wistful remembrance of holidays past. Remember getting that new LEGO set when you were six-years-old? The excitement of unwrapping a new video game? The screech you squealed when you finally got the newest iteration of Ken and Barbie dolls? In fact, it could have been this latter gift that spurred your desire to pursue science, seeing as Barbie herself has pursued over 150 careers in her lifetime , one of the most recent of which was computer engineering. Though her accompanying book I Can Be a Computer Engineer was met with sweeping criticism about Barbie’s reliance on male figures to code her ideas, it nevertheless was an imperative step towards increasing STEM awareness among the highly impressionable toddler set.

 

Quite surprisingly, Ken and Barbie dolls inspire not just receptive to-be scientists, but also the I-already-have-my-PhD-and-receive-federal-funding ones. In this segment of Drosophila Diaries, I’ll explore my favorite fruit fly gene name to date: ken and barbie.

 

You’ve heard it over and over again in your biology classes: Fruit flies provide an exceptional paradigm for studying gene function. They replicate quickly, possess evolutionarily conserved but simplified anatomy and cell behavior, and provide robust genetic tractability. It’s no wonder, then, why scientists in the early 1990’s used Drosophila spermatogenesis as a means to uncover novel genes that govern stem cell identity, mitosis, meiosis, morphogenesis, and cell-cell interactions. Within a single tissue, all of these processes can be empirically observed and probed ad nauseam, providing an unprecedented means to discover new genes (and subsequently name them weird, functionally-specific things).

 

In 1993, Diego Castrillon and colleagues published in Genetics a P-element mutagenesis screen that revealed mutations altering normal tissue function in the fruit fly testis. P-element mutagenesis screens offer some pretty nice incentives that expedite the genetic screening process. It involves a transposable element (those jumpy genes in our genome that plop in and out of place) inserting itself into random genes and disrupting their function by perturbing DNA sequences. P elements can be quickly mapped to genomic locations, used to make new mutant alleles of the gene it settled into, and exploited to clone out surrounding DNA and recover molecular information about its genetic geography.

 

Castrillon et al. generated over 8,000 fly lines that contained P elements plopped into random genomic locations. Of these, over 1,900 flies were screened for altered spermatogenesis; ultimately, they isolated and characterized 83 fly lines in which males couldn’t produce new progeny. These 83 fly lines were subdivided into seven different phenotypic classes, the last of which was a rather peculiar one: “sperm transfer defects.”

 

Male flies with sperm transfer defects essentially had difficulty in the final parts of copulation, the transfer of sperm to females. For example, male parts were sometimes in the wrong place, such as in twig mutant flies where the anal-genital plate was incorrectly rotated. Others, like the pointed mutation, had normal levels of motile sperm stored away but just couldn’t get them from point A to B. These two mutant flies had the right “tools” so to speak, but the final mutant fly in this phenotypic category apparently forgot its toolbox altogether. Flies mutant for one P-element insertion completely lacked external genital. In opening up the male flies, the researchers observed all the internal sexual parts were intact…but where were the outside parts? Whether plagued by holiday nostalgia or not, the scientists knew exactly what to name this new mutant gene: ken and barbie, after the dolls that also do not possess external genitalia.

 

As you’re wrapping up that gift for your brother or sister, niece or nephew, next-door neighbor, or local toy drive, consider two things: (1) how will this gift impress upon the next generation to enter into STEM fields, and (2) how will this gift inspire the hilarious naming of currently undiscovered genes and let scientists leave their comedic mark for decades to come. And if you’re in search for gift ideas for your fellow science enthusiasts, Scizzle has you covered.  ‘Tis the season.

 

10 Must-See Microscopy Images: The Body

 

By Michael Burel

 

As they saying goes: “Beauty is in the eye of the beholder.” No, literally. It’s probably in your eye. At the microscopic level, cellular structures—such as those found in the retina—form breathtaking patterns, taking on color and shapes when combined with fluorescent markers and high-definition capturing techniques. Here are ten must-see microscopy images from the bodies of mammalians to flies.

 

1. Retinal ganglion cells from a mouse retina

Retinal ganglion cells (RGCs)
Josh Sanes, Harvard University
Image courtesy Cell Picture Show.

Retinal ganglion cells (RGCs) relay signals from the rods and cones in the eye to the brain. In this image, RGCs can be seen “pointing” to a single direction and respond best to objects that move in the direction that these cells point.

 

2. Epithelial cells in a developing mouse embryo

Evan Heller, Rockefeller University Image courtesy Nikon 2012 Small World Competition.
Evan Heller, Rockefeller University
Image courtesy Nikon 2012 Small World Competition.

This genetically engineered mouse embryo allows for the visualization of epithelial cells, showing the pattern of whisker placement on the face.

 

3. Crypts and villi of a mouse colon

Paul Appleton, University of Dundee Image courtesy Nikon 2006 Small World Competition.
Paul Appleton, University of Dundee
Image courtesy Nikon 2006 Small World Competition.

This 740-maginified view of the mouse colon was captured using two-photon excitation microscopy. Individual cells making up the villi can be seen protruding out of the screen.

 

4. Utricle from a mouse ear

Jeffery Holt, University of Virginia Image courtesy Olympus BioScapes 2006 Competition.
Jeffery Holt, University of Virginia
Image courtesy Olympus BioScapes 2006 Competition.

The utricle, located deep within the mammalian inner ear, uses small hairs to relay information regarding balance, motion, and spatial orientation.


5. Little skate embryo

Katherine O’Shaughnessy and Martin Cohn, University of Florida Image courtesy FASEB 2013 BioArt Competition.
Katherine O’Shaughnessy and Martin Cohn, University of Florida
Image courtesy FASEB 2013 BioArt Competition.

Researchers in developmental biology turn to cartilaginous fish like skates to understand how complex organisms arise from just a single cell. Little skates are useful in this endeavor because they can develop outside their egg casing, allowing researchers to observe organ formation and development as it occurs.

 

6. Fruit fly ovaries

 

Denise Montell, University of California, Santa Barbara Image courtesy Nikon 2012 Small World Competition.
Denise Montell, University of California, Santa Barbara
Image courtesy Nikon 2012 Small World Competition.

Nearly 85% of cancers derive from epithelial cells that line our organs, and when cancers become aggressive, they can lose their epithelial characteristics and metastasize. Fruit fly ovaries are an excellent model system to study this phenomenon as groups of epithelial cells detach and migrate during egg development.

 

7. Rat cerebellum

Hiroaki Misono, Doshisha University Image courtesy Olympus BioScapes 2006 Competition.
Hiroaki Misono, Doshisha University
Image courtesy Olympus BioScapes 2006 Competition.

This image taken with confocal microscopy shows a section through the rat cerebellum with different neurons and support cell types.

 

8. Blood vasculature from a mouse retina

Michael Bridge, University of Utah Image courtesy  Olympus BioScapes 2012 Competition.
Michael Bridge, University of Utah
Image courtesy Olympus BioScapes 2012 Competition.

In the developing retina, astrocytes (support cells of the nervous system) must establish a framework before blood vessels can grow, much like tracks must be laid down before a train can run. In this three-week-old mouse retina, blood vessels can be seen at different depths: red to green vessels form the primary layer while cyan to purple vessels form the underlying layer.


9. Early brain structures of a developing zebrafish

Michael Hendricks, National University of Singapore Image courtesy Nikon 2007 Small World Competition.
Michael Hendricks, National University of Singapore
Image courtesy Nikon 2007 Small World Competition.

Fluorescent markers reveal details of the midbrain and diencephalon, early structures in nervous system development, in a zebrafish embryo.

 

10. Muscle fibers in a developing fruit fly

Timothy Mosca, Stanford University Image courtesy Nikon 2012 Small World Competition.
Timothy Mosca, Stanford University
Image courtesy Nikon 2012 Small World Competition.

Sacromeres allow muscles to contract and relax, providing a means for locomotion. In this fruit fly larva, sacromeres appear as the thin, striped bands within the red muscle tissue along the body wall. Flying insects have sacromeres that are built with a limited range of motion to assist in wing movement during flight.

 

New Insight Into Breast Cancer Offers Therapeutic Hope

 

By Asu Erden

Triple negative breast cancers are highly aggressive malignancies. They do not express any of the hormone receptors usually used to target chemotherapies to treat this type of cancer and have a high relapse rate after treatment. As such, these cancers can come with a very poor prognosis and insight into their development is therefore direly needed. A study published this month by Chen et al. in the scientific journal Nature dissects the role of the XBP1 protein in the development of triple negative breast cancers. The team of scientists from Weill Cornell Medical College observed that XBP1 levels are higher in triple negative breast cancer cell lines. Of particular therapeutic relevance is their finding that depleting XBP1 leads to reduced tumor metastasis in both a mouse model of triple negative breast cancer and human cell lines derived from such cancers. These findings offer hope for the development of therapies aimed at treating this highly challenging cancer.

 

Cancers have high proliferative rates. This incurs a high energetic cost on cells by requiring the rapid synthesis of proteins. The resulting accumulation of unfolded proteins can in time lead to cellular stress. Studies have shown that the unfolded protein response (UPR) is activated in most breast cancers. The UPR is a cellular stress response mediated by the enzyme IRE1. The role of this enzyme is to cut up the Xbp1 mRNA into its mature form and allow the activated XBP1 protein to translocate to the nucleus. There, XBP1 acts as a transcription factor and allows the expression of a host of genes involved in the UPR.

 

To investigate the effects of anti-XBP1 treatment on cancer relapse, Chen et al. treated a breast cancer mouse model with a combination of XBP1 short-hairpin RNA (shRNA) and doxorubicin (a chemotherapeutic drug). XBP1 shRNA prevent the expression of the XBP1 gene. This combination therapy prevented tumor growth and relapse. Further probing revealed that XBP1 shRNA acts by targeting a specific tumor cell subset – human breast cancer stem cells – known to be involved in tumor relapse. Isolation of this cell population from triple negative breast cancer patients revealed increased levels of activated XBP1. Moreover, the silencing of XBP1 in these mammary gland cells resulted in reduced cell clumps, while overexpression of this gene resulted in increased cell clump formation and resistance to chemotherapeutic drugs.

 

Chen’s team also further dissected the mechanism allowing XBP1 to promote the development of triple negative breast cancers. They unraveled the protein’s involvement in the hypoxia-induced cellular stress response. Hypoxia – a condition characterized by a deficiency in the amount of oxygen reaching cells – is a potent cellular stressor. It is also a central feature of many tumors. The hypoxia-induced factor 1a (HIF1a) is activated during the cellular response to hypoxia and is known to be upregulated in triple negative breast cancers. Chen et al. shed light on the interplay between XBP1 and HIF1a, which was hitherto unknown. They revealed that the two proteins cooperate in targeting specific DNA sequences and that XBP1 increases HIF1a activity. XBP1 therefore allows the hypoxia response, characteristic of cancers, to take place by promoting the cellular responses mediated by HIF1a.

 

The results from this study shed light on the mechanism through which XBP1 contributes to the development of triple negative breast cancers. Of particular note is Chen et al.’s silencing data. Therapies utilizing XBP1 silencing techniques, such as shRNAs, combined with chemotherapies could result in highly successful clearance of these cancers and significantly reduced chances of relapse.

 

Clones In Space, I Have Placed (Infographic)

 

By Brent Wells, PhD

 

Did Lucasfilm Ltd. direct an explosion in cloning efforts at first rumors of the storyline for Episode II, Attack of the Clones? Or did scientist’s unstoppable desire to achieve the impossible instruct the fate of the Empire? We may never know. But the happy coincidence and a recently christened holiday have brought you science in pictures so don’t think about it too much and enjoy.

 

Credit: Brent Wells, PhD
Credit: Brent Wells, PhD.
Click on the image and then expand to full screen.

 

If I’ve managed to assemble this infographic even half as well as I imagine George Lucas can assemble a sandwich, you probably command a decent understanding of the history of cloning technology by now. Like the special effects technologies developed at Industrial Light and Magic (ILM), cloning has advanced from its humble, yet provocative beginnings, into something awe-inspiring and useful at once. Unlike ILM special effects, each subsequent step in the maturation of cloning tech brings something more impressive than before.

 

A new study published just last week in the journal Nature describes the creation of a human, diploid, embryonic stem cell population using SCNT from an adult with Type 1 Diabetes. This is huge for a number of reasons: 1) They were able to use tissue from an adult, which negates any ethical concerns surrounding use of embryonic or fetal tissue. 2) They created diploid cells that can be used in treating human disease. Similar embryonic stem cells were generated in 2011 but were triploid, which means they contained three sets of chromosomes instead of the normal two found in humans, making them non-compatible and therefore inviable for use in disease treatment. 3) The stem cells, cloned from an individual with Type 1 Diabetes, can give rise to the very cells lost due to Type 1 Diabetes, and since they are clones of the affected individual, his/her body will not reject treatment that introduces new cells into their body to replace those lost to the disease.

 

This advancement in cloning technology is a significant step forward in creating stem cell banks that can actually be used in the study and treatment of disease on a case-by-case basis and will extend well beyond Diabetes. It also furthers efforts in the growth of complete replacement organs for those in need of matching donors – after all, there’s no better match for you than you.

 

If you want to learn more about cloning, *waves hand in front of face, uses weird voice inflection* You want to learn more about cloning. You’re going to look into the following resources. I am not the droid you’ve been looking for.

 

Wikipedia, of course

The Basic Science Partnership at Harvard Medical School

The Animal Biotechnology Resource at UCDavis

The Genetic Science Learning Center at the University of Utah Health Sciences

Or just Google it…

 

May the 4th be with you.

Building Better Beer, One Nucleotide at a Time

 

By Brent Wells, PhD

An international team of scientists headed by a group at the New York University Medical Center has created the world’s first eukaryotic synthetic chromosome, meaning they have literally engineered life from its smallest units. Intrigued? You should be.

 

This work, recently published in the journal, Science, was achieved in the common budding yeast, Saccharomyces cerevisiae; the very same little fellow that makes your bread rise and your beer ferment.

 

A chromosome, in case you were wondering, is a continuous grouping of a subset of your genes. All 20,000+ human genes are spread across 23 distinct chromosomes while yeast genes, about 6,000 of them, are spread across 16. The goal of this study was to choose one of those naturally occurring yeast chromosomes and replace it with one synthesized, from scratch, in the lab.

 

How do you synthesize a chromosome? The answer is bit-by-bit-by-bit and with plenty of cheap help.

 

The group started with small, overlapping oligonucleotides, which are very short pieces of DNA – about 70 base pairs in this case. Next, you need an army of undergrads trying to earn an A grade in their Building-A-Genome class, and whose parents are unknowingly paying for your research, to stitch all of these small pieces together into increasingly larger fragments. This is what I imagine building a weave for Rapunzel would be like. Final assembly is completed in the yeast cell where the natural chromosome is replaced, one chunk at a time, with corresponding pieces of synthetic chromosome via a process called homologous recombination.

 

This was not, however, a Gus Van Sant-Psycho-shot-for-shot remake of the original. The chromosome lost a little weight in the process, trimming down to 272,817 base pairs from 316,617. Remarkably, the synthetic yeast were just as viable as the naturally occurring strain, suggesting that there’s a lot of useless DNA floating around in our cells. Among the discarded bits were regions of non-coding DNA called introns as well as transposons. Transposons are DNA sequences that can actually jump around the genome carrying other pieces of DNA with them and which are thought to be a major driving force in evolution.

 

Speaking of evolution, the group also engineered in sequences that would allow them to randomly alter the genome by taking out non-essential genes in a process they call SCRaMbLE-ing. The removal of these genes allows the team to look at the effects of variable-scale genome size reduction on viability. In other words, they can induce a genome ‘scramble’ in millions of yeast cells at once, which will remove different subsets of genes in each, and look at which genes are gone in the ones that survive. This mimics genetic deletion events that can happen naturally during evolution and will help us understand how evolution may occur and the pressures that can lead to the traits it eventually fixes. You can also really speed up a notoriously slow process.

 

This is not the first time a synthetic genome has been attempted, or completed. Groups have had success with viral and bacterial genomes in the past, but this is the first instance of something on this scale. Other groups are currently working on more of the 16 yeast chromosomes with the goal of eventually creating a completely synthetic yeast cell.

 

Beyond the potential to understand mechanisms of evolution and just see if we can actually do it, generation of synthetic organisms have far-reaching commercial potential. Synthetic yeast could be used to generate more efficient bio-fuels, rare medicines for Malaria and Hepatitis and more. And it would be cheap – at least in principle; did I mention they are calling these ‘designer’ chromosomes? I can only assume the synthetic strain was code-named Fendi or Prada.

 

So, should you be worried about ingesting some synthetic yeast during your next trip to Dunkin’ Donuts or Subway? Hardly. Scientists have engineered fail-safes into the synthetic chromosomes that make it impossible for the yeast to live outside of special conditions provided only in the lab. Of course, they did the same thing on Isla Nublar in Jurassic Park and anyone that’s seen Jurassic Park II knows that Jeff Goldblum nailed it when he demanded ‘Nature always finds a way’. But to those alarmist naysayers saying ‘What about the potential for environmental catastrophe?’ Let me offer this recompense: ‘What about the potential for better beer!’

Can a Mutation Protect You From Diabetes?

 

Evelyn Litwinoff

For the first time in diabetes research history, researchers have found mutations in a gene that is associated with a 65% decrease in risk of developing type 2 diabetes (T2D).  What’s even more astounding is that only one copy of the gene has to be mutated to show this protection.  The gene of interest is SLC30A8, which encodes a zinc transporter in pancreatic islet cells.  (A quick brush up on your cellular anatomy: Pancreatic islet cells produce insulin, which the body uses to uptake glucose into cells.  Zinc plays an important role in the uptake, secretion, and structure of insulin.) This study found not 1, not 2, but 12(!) different loss-of-function mutations, all in SLC30A8 and all predicted to result in a shortened protein, that associates with protection from T2D risk.

 

Most of this study is based upon sequencing genes that were previously associated with a risk of developing T2D.  Overall, the authors looked at about 150,000 individuals from various ethnic populations in order to obtain statistical significance for their associations.  Their results are surprising since previous studies had linked mutations in SLC30A8 with an increased risk of T2D.

 

However, this study does not address how a decrease in function of the zinc transporter, named ZnT8, could lead to protection from a disease state.  The authors did conduct one mechanistic-ish experiment, but this was only to see if the mutations in ZnT8 actually affect the activity of the protein.  To this end, the authors overexpressed 4 different mutated versions of ZnT8 in HeLa cells and saw a decrease in protein levels in 2 out of the 4 versions.  Furthermore, they showed that the increased protein degradation could be part of the reason for the observed decrease in amount of protein.  Their main conclusion from these cell experiments show that some of the mutations in ZnT8 result in an unstable protein, which would help us understand how the zinc transporter is not working, but it does not explain why the dysfunctional protein protects from T2D.  Hopefully, this paper will spark others to investigate a mechanism for the associated protection.

 

Currently, Pfizer and Amgen are starting to develop drugs that mimic this mutation to see if they can replicate the protection.  Although a new diabetes drug based on this study could be 10-20 years down the road, this study still makes a big splash in the diabetes research community.

Mitochondrial Clues for a Long Life

 

By Thalyana Smith-Vikos

Biological clocks that can predict an individual’s lifespan more accurately than chronological time alone have been proposed in multiple molecular, cellular and genetic contexts, but a single clock has yet to be identified. Mitochondria, however, have been identified as promising candidates for a biological aging clock in many organisms. Dong and colleagues report that mitochondrial function in Caenorhabditis elegans young adults provides a highly accurate predictive measure of eventual longevity of individual nematodes.

By visualizing quantal mitochondrial flashes, or mitoflashes, in vivo, the authors were able to show that this optical readout was specific to free-radical production and metabolic rate at the single-mitochondrion level. These mitoflashes exhibited a strong correlation with C. elegans aging and had similar attributes in a mammalian system. Mitoflash measurements in pharyngeal muscles peaked during active reproduction and when the first nematodes began dying off. The mitoflash activity on day 3 of adulthood during active reproduction explained up to 59% of lifespan variation. Day 3 mitoflash frequency was negatively correlated with future lifespan of individual C. elegans, and this negative correlation persisted in the face of various genetic and environmental alterations that extend or shorten lifespan. The authors further showed that day 3 mitoflash frequency was due to glyoxylate cycle activity, and they propose that mitochondrial activity not only predicts but also determines lifespan, as the lifespan of long-lived insulin receptor mutants was at least partially explained by decreased mitochondrial production of superoxide.

These findings indicate that mitochondria can function as a biological clock that predicts lifespan of individual C. elegans in various contexts. Importantly, this clock has already begun ticking very early in life, as mitochondrial flashes in early adulthood during active reproduction have been shown to be most potent predictors of future longevity.

 

Microscopy, Mice, and HIV

 

By Elaine To

Monkeys infected with simian immunodeficiency virus (SIV) have been the traditional animal model for the study of HIV pathology. However, SIV does not result in the same immunodeficiency that HIV does, and monkeys are expensive to care for. Mice without immune cells can be engrafted with human immune cells and used instead. The specific model used by Ladinsky et al transfers human fetal thymic and liver tissues along with hematopoietic stem cells. These mice, known as BLT mice, reconstitute human immune cells in significant levels in many tissues, and HIV infection results in T cell depletion.

Ladinsky et al. use a powerful microscopy technique known as cryoelectron tomography in addition to immunofluorescence to understand the characteristics of HIV infection in the small and large intestines in BLT mice. The interior of the small intestine has an upper layer that includes the villi, known as the lamina propria. Between the villi in the lower layer are intestinal crypts, and this is where the majority of HIV viruses were located. Any villi that had evidence of HIV were also adjacent to an infected crypt.

Looking closer, the researchers were able to see individual viruses in the process of budding out of infected cells. It was possible to distinguish between mature and immature viruses based on the differences in internal structures. An examination of the viral pools located outside cells showed that 90% of the pools were mostly mature, but 10% were mostly immature. This is in contrast with previous studies in cell culture showing all viruses found outside cells are mature, indicating a difference in virus maturation or diffusion between cells organized in tissues and cells cultured in vitro.

After some searching, an isolated infected cell was located that was responsible for the production of all viruses in the nearby region. The single cell produced 63 viruses, but the microscopic methods only saw viruses in the same plane as this cell. Regions above and below the cell could not be examined, so the real number of viruses that can result from a single infected cell is likely much more than just 63.

Antibodies targeting CD4 showed that uninfected cells have CD4 on their outer membranes, whereas infected cells have CD4 on their inner endoplasmic reticulum. This supports the previous finding that the HIV protein Vpu causes the internalization of CD4 to prevent newly released HIV viruses from reattaching to the host cell.

There was also evidence of the virological synapse, the phenomenon that happens when a virus budding out of an infected cell immediately contacts a neighboring cell and infects it. Two of the proteins that help bring two cells close together, LFA-1 and ICAM, were found at the cell-cell junction near the actively budding virus.

Lastly, the researchers looked for evidence of the ESCRT proteins, which are known to help release viruses from infected cells. The ESCRT components hCHMP1B, hCHMP2A, and hALIX were found on the thin membranous necks of actively budding viruses. Some budding viruses with thick necks appeared to be in an early stage of budding, and displayed spoke-like projections originating from the virus. These were proposed to be the early components of ESCRT.

Overall, the combination of advanced microscopy with the BLT mouse model revealed new aspects of the process of HIV infection, and showed that conclusions drawn from in vitro cell culture cannot always be assumed to be true in whole animals. Further evidence was also gained for the virological synapse and use of ESCRT proteins to facilitate the spread of HIV within whole organisms.

Backdoor Targeting of the Cancer Causing Protein K-Ras

 

Elaine To

When targeting a specific protein with a small molecule drug in order to treat a disease, scientists often use a molecule that mimics the natural substrate of the enzyme and targets the active site. However, this approach has met with limited success in the case of the oncogenic GTPase K-Ras. GTPases are regulatory proteins that act like binary switches for cellular pathways. In its “on” state, K-Ras is bound to GTP and activates signaling cascades responsible for cell growth, survival, and differentiation. When GTP gets hydrolyzed to GDP, K-Ras is turned “off.” Mutations that prolong the lifetime of GTP when bound to K-Ras, such as the G12C (glycine at position 12 is changed to cysteine) mutant, are highly oncogenic and lead to cancer. The high affinity of K-Ras for GTP and GDP makes drug targeting of the K-Ras active site difficult, but researchers Ostrem, Peters, et al. have discovered an alternate site on K-Ras that can be targeted for cancer therapies.

The researchers set out to find a small molecule that could specifically bind to the oncogenic G12C mutant protein while avoiding the wild type K-Ras by screening a disulfide library, which would be expected to react with the thiol group of the cysteine. Intact protein mass spectrometry revealed which compounds bound to the G12C mutant without targeting the wild type. The two strongest binders were unaffected by the presence of excess GDP, indicating that they do not compete with GDP for binding. X-ray crystallography showed that one of the strong binders was binding in a previously allosteric pocket of K-Ras.

In order to further characterize the novel allosteric site, the researchers examined libraries containing electrophiles, acrylamides, and vinyl sulphonamides for G12C K-Ras binding. Co-crystals of potent binders with K-Ras revealed that the switch-I and switch-II domains of the protein are disrupted, which also disturbs magnesium ion binding. Previously studied mutations in the residues that coordinate the magnesium ion result in a preference for GDP over GTP, thus the researchers tested the compounds for this activity as well. Indeed, exchange assays reveal a shift in K-Ras’s preference from GTP to GDP when the potent electrophiles are bound. Additionally, the compounds can block nucleotide exchange by exchange factors, though EDTA still effectively catalyzes the exchange of GDP for GTP.

It was also noted that the potent compounds occupied a position normally reserved for G60 when K-Ras is active. Known mutants of G60 have impaired binding to partner effector proteins such as Raf. Studies in cell lines show that compound binding impairs the association of K-Ras with Raf. Lastly, in order to show the effectiveness of the identified compounds as chemotherapeutic drugs, the researchers treat various cancer cell lines, some of which contain the G12C mutation. As expected, the cells with the mutation demonstrated significantly decreased viability in the presence of the compounds.

Overall, this is an elegant approach to small molecule drug development that fortuitously revealed a novel regulatory site of K-Ras. Drugs that target this site can be designed specifically for oncogenic mutations, and do not have to overcome the significant barrier of trying to out compete GDP and GTP for binding. The extensive crystal structure and enzymatic characterizations lay the groundwork for further drug development on K-Ras and may open up a whole new class of chemotherapeutic drugs.