Falling in love with bacterial genetics

Marraffini was obsessed with space, astrophysics, and science fiction from a young age while growing up in Argentina. After reading about the advent and promise of recombinant DNA technology in a popular science magazine, his interest shifted toward biology. “During my undergraduate degree in biotechnology in Argentina, I did a lot of DNA manipulation and generated recombinant proteins. This experimental knowledge in molecular biology motivated me to follow a research path,” recollects Marraffini. Because the research opportunities were better in North America compared to Argentina, Marraffini uprooted his young family to pursue a PhD at the University of Chicago.

As part of the PhD curriculum, Marraffini recounts, “I took a class on bacterial pathogenesis and found molecular mechanisms by which bacteria cause diseases fascinating. I found bacteria a great experimental system because many tools were available to mutate and overexpress almost anything. There were also a lot of possibilities to purify proteins of interest using in vitro assays. This is why I fell in love with the bacterial experimental system and ended up joining the laboratory of the course teacher Olaf Schneewind for my PhD.” 

Dissecting CRISPR mechanisms in bacterial immunity

Bacteria are numerous but they are outnumbered by viruses that infect them. CRISPR-Cas is a major immune defense system that evolved in bacteria to fight viruses. Marraffini was interested in how bacteria employ CRISPR mechanisms to interact with and nullify infiltrating DNA and RNA. As a postdoc, Marraffini worked with Eric Sondheimer to experimentally demonstrate for the first time how CRISPR works against conjugative plasmids containing antibiotic resistance. “We showed that CRISPR can prevent the dissemination of antibiotic resistance among bacteria by directly targeting plasmid DNA,” explains Marraffini. This milestone in the CRISPR field was important later for gene editing technology development in mammalian cells. “I collaborated with Feng Zhang at the Massachusetts Institute of Technology. We transplanted a CRISPR-Cas9 system from Streptococcus pyogenes into human hepatocytes and showed that CRISPR cleaves DNA and can be repurposed for gene editing in cells,” shares Marraffini.

Over the years, Marraffini’s group gained mechanistic insights into how CRISPR systems contribute to bacterial immunity. When a phage or a plasmid invades bacteria, the CRISPR system captures a 30- to 40-nucleotides long sequence from the invader DNA called a spacer and incorporates it into the chromosome. This spacer DNA transcribed into the guide RNA gives Cas9—an enzyme that cuts DNA—the target specificity towards invading DNA. This is how bacteria acquire a memory of infection to then fight future infections.

Marraffini also discovered that phage DNA cleavage by Cas9 generates additional DNA fragments, resulting in the acquisition of new spacers for the CRISPR locus. More spacers and guide RNAs against the same-phage DNA are advantageous for bacteria as phages can escape Cas9 cleavage by mutating the target site, offering greater fitness to bacteria. According to Marraffini, “That’s one of our major contributions, showing how spacers acquisition determines infection memory. In addition, we also found that the CRISPR machinery uses free DNA ends, which is a way of diminishing autoimmunity since the bacterial chromosome is circular without a free end.”

Fostering curiosity and boldness

Joshua Modell, Assistant Professor of Molecular Biology and Genetics at Johns Hopkins University School of Medicine, describes his former postdoctoral mentor as “a rare scientist and an intellectual heavyweight who makes the laboratory a stimulating and fun place to do science.” Modell adds, “His ability to interact with and inspire scientists at any career stage, from the greenest summer intern to a long-tenured professor, is what makes him truly special. When I started my postdoc, he explained how much we still had to learn about CRISPR biology and how the work we do could end up in the textbooks. I still try to use that textbook standard with my trainees.”

“My mentors were extremely supportive of my interest in CRISPR despite CRISPR being unknown when I started my academic career,” says Marraffini. He champions the same generosity in his mentorship style, supporting projects his trainees want to pursue. 

“I wanted to investigate a new type of CRISPR that targeted RNA exclusively. No one understood how it worked. While everyone in the laboratory worked on Staphylococcus, I worked on a Listeria strain that naturally carried this RNA-targeting CRISPR system and developed it into a model system,” says Alex Meeske, Assistant Professor in the Department of Microbiology at the University of Washington, who did his postdoctoral training with Marraffini. “He encouraged me to be bold and try new methodologies, even if they were outside his expertise. He taught me to focus, keep my eyes on the prize, and investigate the most significant and testable questions.”

Join us in congratulating Luciano Marraffini, who received the Genetics Society of America Medal at The Allied Genetics Conference 2024 in Metro Washington, DC.

2024 GSA Awards Seminar Series

On September 9, at 1:00 p.m. EDT, Luciano Marraffini will join us to discuss CRISPR-CARF immunity and sacrificing the host for the benefit of the population. Save the date and register here!

Sejal Davla, PhD, is a neuroscientist, science writer, and data scientist with expertise in research in a variety of life sciences. She has more than a decade of experience studying the brain by using cutting-edge methodologies in microscopy, molecular biology, genetics, and biochemistry, and is a motivated storyteller and science communicator.

A new associate editor is joining GENETICS. We’re excited to welcome Bo Zhang to the editorial team.

Bo Zhang
Associate Editor

Bo Zhang is a Professor in Developmental Biology and Genetics at Peking University in China. She received her BS and PhD degrees in Cell Biology from Peking University in 1989 and 1995, respectively, and pursued her post-doctoral training in the Institute of Molecular Biology at University of Zürich, Switzerland. She has been a visiting scholar at University of Wisconsin, Madison, as well as University of California, Los Angeles. Her group is interested in dissecting molecular and cellular mechanisms of vertebrate development through genetic approaches, using zebrafish as the major model with a focus on heart development and regeneration, as well as on developing genome editing techniques in zebrafish based on engineered endonucleases, including TALEN and CRISPR/Cas9.

When female Anopheles mosquitoes take a blood meal from someone with malaria, a tiny Plasmodium parasite enters the mosquito’s digestive tract. That parasite can invade the mosquito’s salivary tissues, so when the insect takes another blood meal, the intruder can slip into the next human host and start a new malaria infection. Malaria is a life-threatening condition that infected 241 million people in 2020 and disproportionately affects vulnerable populations.

To combat the disease, researchers from the University of California, Irvine are developing genetically modified African malaria mosquitoes (Anopheles gambiae) that can’t transmit human malaria, alongside a gene-drive system that can quickly spread those genes and block the spread of the malaria parasite through the population. While this system usually operates with nearly 100 percent efficiency, a small number of mosquitoes will still wind up with mutant alleles that resist the gene drive. Could these mutant alleles sabotage the whole approach? In a paper published in GENETICS, Carballar-Lejarazú et al. looked at this phenomenon and found these mutations didn’t hamper the gene drive in their system.

Malaria-Resistant Mosquitoes

Contributing author Anthony James began exploring genetic methods for controlling vector-borne disease in the mid-1980s. Eventually he exploited mosquito genes that are only turned on in female mosquitoes after a blood meal and linked them with mouse antibodies that protect mice from human malaria parasites.

When these malaria-busting synthetic genes are inserted into mosquitoes, they can’t transmit malaria. And if it mates with a regular mosquito, the beneficial gene will be inherited like any other gene, gradually building up in the mosquito population. But what if that process could happen faster?

That’s when CRISPR gene editing technology hit the scene. “It seems like overnight when you work 10 or 15 years on something to make it work and then something new comes along and—in less than a year—you have it working,” says James.

Gene editing operates on the germline—the cells that will eventually become sperm or eggs—by snipping the normal chromosome and inserting the new sequence, in this case, the malaria fighting gene. In male mosquitoes, this works so well that each male passes on the new gene to nearly 100% of its offspring.

It’s a bit more complicated in female mosquitoes because egg cells are massive compared with sperm. When the system snips the normal chromosome and inserts the synthetic sequence, the second chromosome may be too far away to trigger the repair mechanism that sews the cut chromosome back up while including the system. That means there’s a chance the snipped chromosome will just stick itself back together—called nonhomologous end joining—possibly resulting in a mutant allele that resists the gene drive.

Exploring Gene Drive Mutations

To figure out if those mutant alleles could pose a problem for the gene drive system, the researchers linked the system to a somatic gene for eye color and marked it with a fluorescent protein. Then, the team performed various crosses to see how the genes passed on to future generations. Non-mutant progeny had black eyes (before adulthood) while those with the mutant allele had pink eyes. And all the progeny carrying the gene drive had eyes that fluoresced blue under light.

To make things a bit more complicated, some progeny were mosaics, with a mix of alleles and more complex eyes, but since the eye color gene isn’t part of the germline—it won’t pass on to the next generation—most of those mosaic mosquitoes still passed on the gene drive.

In the lab, about 25 percent of the first generation of progeny received the gene drive. By the fourth generation, the entire population had fluorescent blue eyes—meaning none of those mosquitoes could transmit malaria.

“Four generations is sufficient,” says James.  “That’s well short of one transmission season.”

James is quick to point out that this isn’t a magic bullet for malaria and there is more research to be done. There are also many thorny issues and debates for scientists and the broader community to work through before everyone is comfortable deploying gene drive mosquitoes in the wild. But James is hopeful the project to which he’s dedicated so many years may one day help ease the malaria crisis.

“There was a famous scientist who said a new idea doesn’t take hold because you change people’s minds; it takes hold because there’s a whole new generation of people that have grown up hearing about it,” says James. “I wish it was a little faster, but we’ll do our part, and hopefully people will take it up. It may not be me, but we have something to hand off.”

CITATION:

Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae

Rebeca Carballar-Lejarazú, Taylor Tushar, Thai Binh Pham, Anthony A James

GENETICS

2022: iyac055

https://doi.org/10.1093/genetics/iyac055

Melissa Mayer is a freelance science writer based in Portland, Oregon.

Sydney Brenner famously noted that progress in genetics “depends on the interplay between new techniques, new discoveries, and new ideas, probably in that order of decreasing importance.” In the 40 years since those words were recorded, new techniques have propelled the field of molecular biology to heights barely imagined at its inception.

This year’s recipient of GSA’s Edward Novitski Prize, Feng Zhang of the McGovern Institute for Brain Research and the Broad Institute, proves that Brenner’s adage requires an addendum: in many cases, the process of developing new techniques draws on new ideas and new discoveries. Zhang’s ideas and discoveries paved the way for two revolutionary techniques that have become indispensable to molecular biologists: optogenetics and CRISPR.

“He revealed his brilliance from the very beginning,” says Karl Deisseroth of Stanford University, who was Zhang’s graduate advisor. “The technology we developed in my lab, called optogenetics, requires the convergence of several different streams of technology innovation, and Feng was really central to all of those streams.”

Optogenetics uses light-activated ion channels, called opsins, to create an electrical signal. By engineering brain cells to produce opsins, researchers can then activate just those cells by exposing them to light. The first successful demonstration of optogenetics used a protein from algae called channelrhodopsin, which responds to blue light and activates neurons by moving sodium ions into the cell.

Zhang’s key contributions helped make the technology broadly useful for a variety of experimental uses. First, he discovered new opsins, including the first red light-activated channelrhodopsin, and halorhodopsin, an opsin that transports chloride ions rather than sodium ions. Halorhodopsin enables selective silencing of neuron activity, adding incredible versatility to the optogenetics system. He also developed a viral delivery system that enabled cell-type specific targeting of opsins.

“You can make all the opsins you want, but if you can’t target them in a versatile and generalizable way to specific kinds of cells, you actually haven’t really done much,” Deisseroth says. “That was extremely difficult, and it wasn’t clear how to do that in a generalizable way at the time. Feng developed a very elegant viral targeting strategy.”

The Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. Zhang’s contributions to optogenetics demonstrate plenty of intellectual ingenuity, and then he moved on to an equally impressive tool: CRISPR.

“I got interested in genome editing because of wanting to solve this problem for optogenetics,” Zhang recalls. He began by trying to customize zinc finger nucleases to enable cell-type specific expression of different opsins, but designing a different zinc finger for each target gene was laborious. Next, he worked with TALE nucleases, which were easier than zinc fingers but still time consuming. “Nevertheless, I was excited about this gradually making the system easier and easier to use,” he says. “Then, as I was working on TALEs and teaching my students to use it for their projects, I went to a talk, and I learned about CRISPR systems.”

CRISPR is an adaptive bacterial immune system that allows the cell to recognize and attack viral nucleic acids. Zhang realized that CRISPR nucleases could be the answer to the problem he’d been working on, and he set about developing them for use in mammalian cells.

“This ability to redirect where to target in the DNA without having to change the protein is very powerful,” he says. Zhang refocused his efforts from TALEs to CRISPR, specifically, the Cas9-containing systems. He engineered Cas9 to function in human cells, a watershed moment for genome editing technology.

To expand the utility of CRISPR, just as he’d plumbed nature’s rich toolbox for new types of opsins, he went looking for other CRISPR enzymes in various bacterial species. “Nature is very amazing — it evolved everything that is on this planet,” he says. Using this approach, he discovered that Staphylococcus aureus made a smaller Cas9, which is more amenable to viral delivery, a key aspect of in vivo genome editing.

Over the next two years, he published 22 papers reporting major advances in CRISPR/Cas9 editing. He showed how to dramatically reduce off-target cutting by Cas9 and developed a method of whole-genome loss-of-function CRISPR screening, a powerful tool for gene identification that “will have enormous ramifications in biomedical research,” writes Bert Vogelstein, Director of the Ludwig Center for Cancer Genetics & Therapeutics at Johns Hopkins and one of the scientists who nominated Zhang for the award.

“Dr. Zhang has done, and continues to do, fantastic work,” Vogelstein says. “Through his sharing ideas and materials with the research community, he has enabled numerous laboratories to make major scientific advances.”

More recently, Zhang has discovered new CRISPR enzymes that behave differently than Cas9, including Cas13, which targets RNA. Using Cas13, Zhang helped develop a nucleic acid test called SHERLOCK (“specific high sensitivity reporter unlocking”), which was used to create rapid point-of-care SARS-CoV-2 tests.

As a mentor, Zhang emphasizes collaboration to advance science by combining different people’s expertise. “It also makes doing research more fun,” he says. “You make many more friends from around the world. That is one of the most valuable things that I get from working on these problems.”

He encourages students to be curious about everything and find something that excites them. “That’s the best way for people to be creative and really exceed even their own imagination,” he says. “Once a student finds that passion, then as a mentor my job is to support them with everything I can and help them develop their idea, both scientifically and also teaching them how to engage in meaningful and rewarding collaborations with other people.”

Zhang’s work has previously been recognized with the Alan T. Waterman Award from the National Science Foundation (2014), the Canada Gairdner International Award (2016), the Tang Prize (2016), the Albany Medical Center Prize (2017), the Lemelson-MIT Prize (2017), and the Richard Lounsbery Award from the National Academy of Sciences (2021), among others.

“He’s an incredibly humble guy,” Deisseroth says. “One of the nicest guys you’d ever meet, and a great collaborator.”

Watch Feng Zhang’s GSA Award Seminar: “RNA-Guided DNA Insertion with CRISPR-Associated Transposases”

The Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. The prize honors scientific achievement that stands out from other innovative work, that is deeply impressive to creative masters in the field, and that solves a difficult problem in genetics. It also recognizes the beautiful and intellectually ingenious experimental design and execution involved in genetics scientific discovery.

A better way to make endogenous reporters in C. elegans

CRISPR systems for gene editing have revolutionized biological research, but the method still has limitations. While it is usually straightforward to delete parts of the genome using CRISPR, large insertions can be a challenge. This has been the case even for the nematode Caenorhabditis elegans, one of the most established model organisms. But now, work published in GENETICS by Vicencio, Martínez-Fernández, Serrat, and Cerón has produced a more effective way to use CRISPR to insert longer stretches of DNA into the nematodes’ genomes.

A method for adding long DNA fragments is essential because many genes of interest, including important fluorescent reporter genes, are too long to be effectively inserted using existing methods. In fact, the team embarked on the work after attempts to insert a gene into the nematodes using another CRISPR-based technique repeatedly failed—a problem also reported in at least one other publication. In contrast, they found that their method, called Nested CRISPR, could efficiently add segments of DNA up to 792 base pairs long. They also achieved insertions of 927 base pairs, although the efficiency was lower.

In their method, the gene is inserted in two CRISPR-based steps. First, a short fragment with nucleotides from each end of the gene is inserted into the target site. Next, this fragment is replaced by the full-length insertion via homology-directed repair.

Their results mean that when insertions of a few hundred base pairs are needed, Nested CRISPR is a viable alternative to current methods involving extrachromosomal or randomly inserted DNA. The Nested CRISPR technique may even be broadly applicable to other organisms, particularly through the authors’ one-shot approach to achieve the two editing steps in a single injection.

It’s not completely clear why this group and others have had difficulty reproducing the level of efficiency reported for an existing CRISPR-based method for inserting DNA segments of this length into C. elegans. Slight differences in reagents among labs may be partially to blame for the lack of reproducibility of some laboratory methods, including those used for genome editing, but the authors of this study believe that won’t be an issue in the case of Nested CRISPR because all the reagents are commercially available and affordable. The availability of these premade reagents may also make it easier for researchers with less experience in gene editing (or molecular cloning in general) to perform the technique, allowing them to pursue projects that they otherwise may have avoided. The group has called for the C. elegans community to come together to evaluate the utility of methods such as theirs for inserting long stretches of DNA—which may become even more important as the field continues to hurtle forward.

CITATION:

Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
Jeremy Vicencio, Carmen Martínez-Fernández, Xènia Serrat, Julián Cerón
GENETICS 2019 211(4): 1143-1154; https://doi.org/10.1534/genetics.119.301965
https://www.genetics.org/content/211/4/1143

Wild yeast aren’t picky about their mates. For Saccharomyces cerevisiae, setting the mood is as simple as providing an abundant supply of nutrients, which prompts each yeast cell to search for another of the opposite mating type. If a lonesome yeast cell can’t find a suitable partner, it’s no problem—it can alternate between mating types, if needed, each cell division.

In contrast, lab yeast can’t pull off this trick; they carry deletions or other disabling mutations in parts of their genome needed for mating-type switching. Their inability to switch helps prevent the yeast from inconveniently changing types in the middle of an experiment, but it also creates a lot of extra work for geneticists when they’re trying to construct new strains while maintaining strict isogenicity. Now, researchers have developed a CRISPR/Cas9-based method to rapidly and efficiently switch mating types in a single cotransformation.

Mating-type switching is essential for constructing strains that are genetically identical except for the mating-type locus. These isogenic haploid strains are useful because they can mate with each other to form a diploid cell with two identical copies of the genome. And since diploid cells differ in many ways from their haploid equivalents, it’s often important to study genetic phenomena in both.

The ability to induce yeast to switch mating types is a particularly vital tool in the assembly of Sc2.0: a completely synthetic yeast genome. Creating and subsequently studying this genome could answer deep questions about biology—like how far we can go in modifying a eukaryote’s genome while still keeping the organism alive.

Completing this massive project, which is led by some of the authors of this study along with their collaborators around the world, will require many mating-type switches to consolidate the final synthetic chromosomes in a single strain. Switching the mating types of lab strains used to require a process with many steps, including manual dissection of spores using an exceptionally fine needle under a microscope. Having to rely on this classic but laborious process would slow the progress of the Sc 2.0 project, so the faster and easier CRISPR-Cas9-based technique offers a major advantage.

Although construction of the synthetic yeast genome isn’t yet in its final stages, many other yeast geneticists stand to benefit from the group’s speedy new technique, which would also be useful for building isogenic pairs of strains and even building isogenic tetraploids. Plus, freeing up geneticists from tedious tasks gives them more time to dream up schemes for extraordinary advances—like a whole genome built from scratch.

CITATION:

Xie, Z.; Mitchell, L.; Liu, H.; Li, B.; Liu, D.; Agmon, N.; Wu, Y.; Li, X.; Zhou, X.; Li, B.; Xiao, W.; Ding, M.; Wang, Y.; Yuan, Y.; Boeke, J. Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae.
G3, 8(1), 173-183.
DOI: 10.1534/g3.117.300347
http://www.g3journal.org/content/8/1/173

Why study human diseases in frogs? For starters, 79% of genes implicated in human disease have orthologs in the African clawed frog Xenopus laevis. Frogs also produce hundreds of embryos that can be grown in a dish, meaning they can be manipulated in ways that are impractical on a large scale in mammals. For example, scientists can microinject specific cells in the developing frog embryo as a way to alter the genes in particular organs in the adult. This is particularly useful since disruption of many important developmental genes is lethal, which prevents detailed investigation into these genes’ functions.

Though frogs as a model system allow for scales not easily achieved in other species, there are limitations to the genetic tools available. Current knockout techniques, like the use of morpholinos, are expensive and often toxic. In a report in GENETICS, DeLay et al. showed how CRISPR technology can be used for tissue-specific gene editing in X. laevis.

CRISPR is a gene editing technique which is quickly revolutionizing many aspects of genetics, and DeLay et al. used it to knock out lhx1 in the kidneys of developing frogs. Lhx1 is important for development of the kidney, head, spine, and other structures in both frogs and mice. Embryos lacking functional lhx1 rarely survive, and if they do, they have severe defects. The researchers used CRISPR to functionally knock out lhx1 in a targeted manner by microinjecting different cells in the frog embryo. For example, injection of one of the cells in a  two-cell embryo knocks out lhx1 in half of the frog, leaving the other half undisturbed. These frogs then develop with one normal, unaffected kidney and one deformed kidney. This method will be especially useful for genetic screens because the unaffected kidney serves as a near-perfect internal control.

The researchers hope that this relatively inexpensive technique will be used to further study the role of different genes in organogenesis and development. It will also make frogs a better model for studying human disease, since CRISPR sidesteps many of the problems of other knockout methods. Discoveries in frogs can help us better understand what the human counterparts of these genes do and, hopefully, pave the way for advances in combating human disease.

CITATION
Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9

Bridget D. DeLay,  Mark E. Corkins, Hannah L. Hanania, Matthew Salanga, Jian Min Deng, Norihiro Sudou, Masanori Taira, Marko E. Horb and Rachel K. Miller

Genetics February 2018, 208: 673-686;

https://doi.org/10.1534/genetics.117.300468 

http://www.genetics.org/content/208/2/673

It’s surprisingly common for babies to be born missing one or both kidneys; an estimated one in one thousand babies are born with a single kidney. Called renal agenesis, this condition is fatal if both kidneys are missing, and having just one can also lead to serious health problems like hypertension and early renal failure. In the September issue of GENETICS, Brophy et al. show for the first time that renal agenesis in humans can be caused by disruptions in the retinoic acid receptor pathway. They used whole exome sequencing in two affected families to identify a causal mutation and applied innovative CRISPR mutagenesis in mice to confirm their findings.

Two unrelated families from Iowa and Denmark each had multiple cases of renal agenesis. For both families, the researchers identified potentially causal mutations by comparing the whole exome sequences of several affected and unaffected family members. The gene GREB1L carried harmful mutations in both families: a missense SNV in one and a deletion interrupting a splice site in the other. Further sequencing confirmed that all affected individuals carried the mutated gene copies.

GREB1L is a cofactor for retinoic acid receptors that until now has never been implicated in mammalian kidney development—let alone renal agenesis. To confirm its effect, the researchers obtained a zebrafish mutant for the corresponding gene. Fish homozygous for the mutation showed abnormal early kidney development and died before reaching maturity. Knockdown treatment that decreased GREB1L expression in genetically normal fish had similar results, suggesting that GREB1L was indeed the gene causing the kidney developmental problems.

The final confirmation came from CRISPR-mediated mutations which were generated in F0 mice, eliminating the need for performing genetic crosses. Brophy et al. replicated the GREB1L mutation found in the Iowa family and generated mice with a variety of kidney development phenotypes spanning the range observed in the family. This suggests that there is developmental flexibility in how much GREB1L expression is needed to make one or two healthy kidneys. Furthermore, the use of CRISPR to generate mice that mirrored human phenotypes demonstrate how this technology can be used to quickly model idiosyncratic human mutations to better understand the causes of conditions like renal agenesis.

CITATION:

A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans

Patrick D. Brophy, Maria Rasmussen, Mrutyunjaya Parida, Greg Bonde, Benjamin W. Darbro, Xiaojing Hong, Jason C. Clarke, Kevin A. Peterson, James Denegre, Michael Schneider, Caroline R. Sussman, Lone Sunde, Dorte L. Lildballe, Jens Michael Hertz, Robert A. Cornell, Stephen A. Murray and J. Robert Manak

GENETICS September 1, 2017 207: 1 215-228; https://doi.org/10.1534/genetics.117.1125

http://www.genetics.org/content/207/1/215

In the life of a butterfly, color is crucial. Color helps these flashy insects attract mates, avoid being spotted, or even signal to predators that they would make a bad meal. On the cover of the March issue of GENETICS is a close-up view of Junonia coenia, a butterfly with stunning blue eyespots on its wings. This species, along with three other types of butterflies, was the subject of research by Zhang et al. into the ways butterfly wings get their eye-catching hues.

In Junonia coenia, mutations in pale result in these barely-colored eyespots by affecting the black pigment beneath the blue scales, which normally absorbs excess light and intensifies the blue color. Photograph by Arnaud Martin.

The goals of the study were to find and characterize genes that might play a role in wing color development, to determine whether previously described genes that affect color in other insects and  unknown candidate genes play roles in butterfly wing coloration, and to figure out the mechanisms behind these genes’ effects on wing color. As well as providing insight into the complex ways butterflies generate their colors, studying pigmentation in butterflies has the unique benefit of an easy readout of a gene’s effect, since the wing patterns can be seen by eye and enhanced by low-magnification microscopes.

In search of genes that contribute to pigmentation, the researchers looked for genes that significantly changed expression in the wings during development, indicating that they might play a role in color formation. To pare down this starting list of over 2,000 genes, they applied stringent screening criteria that narrowed their focus onto just 53.

Close-up of scales in the wing eyespot focus of the wild-type Junonia coenia. Photograph by Arnaud Martin.

Many of the genes they found to affect coloring in butterfly wings are also known to contribute to coloring in other insects, and in many cases, the mechanism behind the genes’ effects on color seemed similar between butterflies and other insects. One of the genes they investigated, called pale, is associated with a light-colored phenotype in flies. Mutating pale caused marked defects in pigmentation in the butterflies’ wings, making their normally vibrant-blue eyespot patterns seem dull. But interestingly, the blue scales themselves were intact—it was the loss of the matte black background, which normally absorbs excess light of other wavelengths, that resulted in the loss of blueness.

That the black background is important is consistent with some recent biophysical research on color formation in butterfly wings. This finding also highlights the importance of increasing the number of model organisms we study for gaining insight into the functions of genes—which, with the advent of CRISPR/Cas9 genome editing, is now a much more feasible goal.

Wild-type Junonia coenia. By Vicki DeLoach via Flickr.

CITATION:

Zhang, L.; Martin, M.; Perry, M.; van der Burg, K.; Matsuoka, Y.; Monteiro, A.; Reed, R. Genetic Basis of Melanin Pigmentation in Butterfly Wings.
GENETICS, 205(4), 1537-1550.
DOI: 10.1534/genetics.116.196451
http://www.genetics.org/content/205/4/1537

When geneticist Rob Unckless took his son to Lego Club at the local library, he was not expecting to start a new collaboration. The result is the striking piece of science-inspired art that graces the cover of the February issue of GENETICS.

Created by artist Kent Smith, “Attack of the 50 Foot Mosquito” was inspired by a paper by Unckless and his colleagues. The study examines a more subtle threat than a rampaging giant: the potential evolution of resistance to gene drives. Gene drives are a burgeoning new technology that use CRISPR-Cas9 genome editing to alter the genomes of an entire population. Cas9 is an enzyme that, when introduced into a cell, makes cuts in DNA that are then repaired through the endogenous homology-directed repair pathway. This repair process can be co-opted to change the final sequence; if a DNA sequence is introduced that carries the desired edit along with some homology to the cut DNA, it can serve as the template for repair. To turn this editing process into a gene drive, Cas9 and the guide RNA are also inserted permanently into the genome, so theoretically every heterozygote will automatically undergo this process. After the first transformation, the gene drive is self-propagating. This technology has been suggested as a way to control populations of disease vectors, like mosquitoes.

Though this technology is exciting and new, selfish genetic elements are definitely not. There are a multitude of natural examples of elements that manipulate their way into more than half of an individual’s offspring. And notably, many of these natural driving elements are associated with genetic suppressors that prevent their selfish activity and restore equal transmission of both alleles. In their paper, Unckless and colleagues discuss potential mechanisms of resistance to gene drive, including new mutations and accidental by-products of the repair process involved in gene drive. They use mathematical modeling to show the probability that resistance spreads through the population is very much dependent on the frequency at which resistant alleles arise in the first place. This study shows much care and study is needed to more effectively use gene drives in the wild.

This stunning cover was born when Unckless had just moved to Lawrence to begin an assistant professor position at the University of Kansas. He took his son to the Lego Club at the library; Kent Smith, a local artist who teaches in the School of Architecture and Design at KU and  specializes in science fiction-inspired artwork, was also there with his son. “I struck up a conversation with Kent,” says Unckless, “We talked about what I do and what he does, and decided it would be fun to collaborate on something.” When the manuscript was accepted by GENETICS, they decided to do a piece to submit as a potential cover.

“I loved being able to provide a fun visual for all of this amazing big brain science!” says Smith. “One of my favorite things about design and illustration is getting to research and learn about new subjects for each piece.” Unckless and Smith discussed what the paper showed about gene drives, and then Smith came up with a way to illustrate the evolution of resistance metaphorically. “Nature’s response to the gene drives comes in the form of a giant monster mosquito rampaging through a city,” he explains. The piece is a reference to the iconic poster for the 1958 science fiction movie Attack of the 50 Foot Woman. Unckless saw rough sketches and gave suggestions about things like mosquito anatomy and the mosquito-borne Zika, dengue, chikungunya, and yellow fever viruses swirling in the clouds of smoke and destruction. A tiny Andy Clark can also be seen fleeing destruction on his bike.

“I was lucky to have such a rich subject matter,” says Smith. “Collaboration and dialogue with Rob was very inspirational and allowed for some great brainstorming.”

The final product of this meeting between art and science is a tongue-in-cheek commentary on the potential perils of gene drive gone awry. Though rampaging giants are unlikely to result, scientists should carefully consider the consequences of this potentially powerful technology.

Unckless, R. L., Clark, A. G., & Messer, P. W. (2017). Evolution of resistance against CRISPR/Cas9 gene drive. GENETICS. 205(2):827-841. DOI: 10.1534/genetics.116.197285

http://www.genetics.org/content/205/2/827

More of Kent Smith’s artwork can be found here:

www.smittytown.com

https://www.facebook.com/KentSmithIllustration/

https://www.instagram.com/smittytownart/

https://society6.com/smittytown