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

Biolistic genetic transformation in C. neoformans produces few off-target side effects.

While genome editing is a staple of genetics research, there remains anxiety about unintended side effects of genetic transformation, one of the most common longstanding genome-editing techniques. Some researchers fear that the process of introducing exogenous DNA into a cell may cause unwanted mutations, adding confounding variables to their experiments—but others aren’t content to accept this lore.

In G3, Friedman et al. report their study of the off-target effects of transformation in the common fungal pathogen Cryptococcus neoformans. They created 23 new strains using biolistic transformation, a standard procedure for this organism that involves shooting gold beads coated with DNA into cells, to add a marker to a neutral site in the strains’ genomes. They then sequenced the genomes of these new strains. Across all 23 strains, they found only four point mutations; of these, just one changed an amino acid in the encoded protein. They also found one case of insertion of a second, partial copy of the drug resistance marker.

They used the same transformation method to create more than 100 strains, each with a single gene replaced by a marker gene. By carrying out RNA-Seq on this group, they identified six strains that expressed the marker at unusually high or low levels. On average, these outlier strains had 1.67 off-target point mutations, and three of them (50%) carried multiple copies of the marker. The greater number of mutations in these six strains compared to the first set of 23 likely reflects selection for mutations that compensate for the genes the researchers replaced. Nonetheless, the overall number of off-target effects was still low, and the authors write that the mutations would be unlikely to have consequences as drastic as deleting the intended gene would. Therefore, they argue, effects observed when a gene is deleted using this protocol are likely most often due to the deletion and not to off-target effects, although additional confirmation of any deletion’s effects is still prudent.

The study illustrates the importance of testing conventional wisdom, and it will be important to investigate whether these findings apply to other species used in research and other transformation techniques. In the process of conducting this study, the researchers also sequenced a frequently used C. neoformans laboratory strain’s genome—a vital resource because this fungus is estimated to kill hundreds of thousands of people each year.

CITATION:

Unintended Side Effects of Transformation Are Very Rare in Cryptococcus neoformans
Ryan Z. Friedman, Stacey R. Gish, Holly Brown, Lindsey Brier, Nicole Howard, Tamara L. Doering and Michael R. Brent
G3: GENES|GENOMES|GENETICS 2018 8: 815-822; https://doi.org/10.1534/g3.117.300357
http://www.g3journal.org/content/8/3/815

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

Thanks to more than a hundred years of working with Drosophila melanogaster, geneticists have many powerful tools for precisely manipulating its genes. It has also become a model system for studying speciation and molecular evolution together with the other members of the melanogaster species group: D. simulans, D. mauritiana, D. yakuba, and D. santomea. However, evolutionary genetic studies have been hampered by an inability to make transgenic lines within the less well-studied species. In the April issue of G3, Stern et al. present a panel of new transgenic strains of these species designed to make fine mapping and functional genetic studies possible. These resources provide new opportunities for unraveling the genetic mechanisms of speciation and evolution.

Transposable elements are selfish genetic elements that insert themselves into new genome locations. This natural machinery has been adapted by scientists to introduce carefully designed DNA constructs into genomes. Stern and colleagues used the piggyBac transposon system to insert a fluorescent reporter gene into hundreds of random locations in the genomes of five different Drosophila species. They mapped the location of each insertion and identified the unique insertions that could be maintained in homozygotes, resulting in 184 D. simulans lines, 122 D. mauritiana lines, 104 D. yakuba lines, and 64 D. santomea lines. Each line has an insertion in a unique genomic location coupled to a fluorescent reporter gene whose expression can be easily detected in the eyes with a microscope. The huge collection of unique locations will make fine mapping in these species much easier.

Coupled to many of the insertions is a landing site that can be used to add new insertions via plasmids carrying a suitable targeting sequence. The authors tested the integration efficiency at landing sites in unique genomic locations and identified those with high efficiency that could be maintained as homozygotes. They also tested the effects of insertion location on expression by transforming a fluorescent reporter gene linked to an enhancer of even-skipped, a developmental gene in D. melanogaster with well characterized expression patterns. In four of the five species tested, at least one strain showed the expected patterns of expression, suggesting that any gene of interest transformed into these sites will not be subject to ectopic expression from location effects. Additionally, they used CRISPR/Cas9 gene editing to knock out expression of the fluorescent eye reporter in a number of strains. Since strains still have the landing site in known locations, they can be used to examine expression of genes of interest in the eye without interference from the reporter.

Noni fruit is toxic to most other fruit flies, but <i>D. sechellia</i> loves it. Photo by Carmen via Flickr.

The fly species of D. melanogaster species group are notable not just for their close relationship with the famous D. melanogaster, but they also make excellent evolutionary models in their own right. D. melanogaster and D. simulans are cosmopolitan species that live wherever humans do, but the other members of the group are found only on islands off the coast of Africa. D. sechellia is a member of the group that feeds primarily on a fruit toxic to most other animals. Despite the adaptive differences that come with these divergent lifestyles, many of these species can still hybridize, making laboratory studies aimed at dissecting these differences possible. The transgenic lines presented in this paper will make studies on speciation and adaptation in these lineages more powerful and accessible than ever before.

Stern, D. L., Crocker, J., Ding, Y., Frankel, N., Kappes, G., Kim, E., Kuzmickas, R., Lemire, A., Mast, J.D. & Picard, S. (2017). Genetic and Transgenic Reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea, and D. virilis. G3: Genes, Genomes, Genetics, 7(4), 1339-1347.

http://www.g3journal.org/content/7/4/1339

Fruit fly larvae have one goal: eat as much as possible. After the tiny worm-like larvae hatch from eggs embedded into the flesh of rotting fruit, they eat their way out. After days of gorging, they find a good spot to pupate and then emerge as adults. Fruit flies cannot grow after this transformation, however, so their body size is fixed by their larval feeding success. Since greater adult body size is associated with increased fitness, those first feasts are critically important. In a paper published in the February issue of GENETICS, Allen and colleagues use a precise knockout to show that multiple feeding-related traits in fruit fly larvae are controlled by a single gene.

The gene foraging encodes a protein kinase that has two naturally occurring alleles —the rover and sitter alleles. Larvae with the sitter allele do not travel as far as those with the rover allele, and they differ in other food-intake related traits. However, the lab strains that carry the rover and sitter alleles differ across the entire chromosome that carries foraging, so these differences could be caused by variation in other genes physically linked to foraging. To precisely determine the phenotypes affected by the gene, Allen and colleagues generated a precise knockout using homologous recombination, enabling them to compare larval behavior in lines that differed only by this single gene. They also made a rescue construct containing the foraging sequence, which they introduced into the knockout to restore the wild-type phenotype. Finally, they overexpressed foraging by inserting this rescue construct into the wild-type background. Using larvae with these three genotypes, they quantified the effect of a range of foraging expression levels.

The larvae that totally lacked foraging expression survived to pupation at a normal rate but failed to successfully metamorphosize into adults. A lack of foraging expression also affected feeding phenotypes. The authors tested whether larvae with the three genotypes  differed in the how far they traveled through their food and found that those with the highest foraging expression traveled the most. They also found that higher expression of foraging correlated with consuming more food, though the larvae’s triglyceride content decreased with foraging expression. Clearly, this gene plays a critical role in several different feeding and metabolism related phenotypes.

The authors suggest that foraging has the ability to control multiple phenotypes because of its transcriptional complexity;foraging has four transcriptional promoters and 21 different isoforms. It is currently unknown how the different isoforms are expressed or what their specific roles are, but the authors propose that tissue or developmental stage specific expression of functionally different transcripts could allow this single gene to affect phenotypes as different as feeding behavior and fat metabolism.

http://www.genetics.org/content/205/2/761

Allen, A. M., Anreiter, I., Neville, M. C., & Sokolowski, M. B. (2017). Feeding-Related Traits Are Affected by Dosage of the foraging Gene in Drosophila melanogaster. GENETICS, 205 (2) : 761-773. DOI: 10.1534/genetics.116.197939

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

Efforts to engineer genomes in wild populations have huge potential for good—but the real world is more complicated than the lab.

Until now, humans have never been able to seriously consider how to cheat evolution. But now that the CRISPR/Cas9 system has made genome editing easy and efficient, it might be possible to manipulate the genomes of entire wild populations—and even to force the spread of genes lethal to their carriers.

Although the risks and ethical concerns of this proposal have sparked debate, being able to control wild populations could have massive benefits, including eradicating devastating human diseases. But while researchers are able to create all types of drives in the lab, the limits on gene drive’s power in the wild haven’t received as much scientific and media attention. Fundamental questions remain unanswered. How will gene drives spread? How—and when—could resistance arise? Spurred by its potential to do good and to do harm, geneticists are racing to understand how gene drive might work—and how it might fail—in the wild.

Population Engineering

In principle, using CRISPR/Cas9 to drive a gene into a population is beautifully simple. Select a target site in the genome to be modified. Deliver to the host’s cells a template consisting of the DNA sequence to be inserted flanked by sequences homologous to the target site, along with a gene encoding the DNA-slicing enzyme Cas9 and a sequence coding for an RNA that guides Cas9 to the right DNA to cut. The Cas9 produced from this construct will chop the host’s genome at exactly the target site specified by the template.

When the cell tries to repair the cut, it often relies on DNA with a matching sequence from the homologous chromosome. But during this process, called homology-directed repair, the cell sometimes mistakes the gene drive template for the homologous chromosome. When the cut is repaired this way, the cell is tricked into including the entire gene drive sequence, cementing a permanent copy into the genome. Now the patched-up chromosome becomes a template itself: Cas9 can snip the normal copy of the chromosome and insert the drive there, too. Heterozygotes for the drive can then pass on the foreign sequence to their offspring as if they were homozygotes. Hypothetically, this would make it possible to engineer an entire wild population using a precise genome edit.

In this depiction of gene drive, a circular DNA molecule called a plasmid is inserted into cells. The plasmid contains a gene for the DNA-cutting enzyme Cas9, a sequence encoding a guide RNA (gRNA) that defines the sequence Cas9 will cut and a gene to be inserted at the cut site (payload gene). These sequences are flanked by homology arms (H1 and H2) that match up with DNA surrounding the cut sequence in the normal chromosome, allowing the whole DNA cassette on the plasmid (H1, Cas9, gRNA, payload gene, H2) to serve as a template for repairing the cut chromosome. Then, the repaired chromosome acts as a template itself when Cas9 cuts the homologous chromosome, so both copies of the chromosome in the cell contain the DNA cassette. Thus, the progeny will inherit the inserted sequence (almost) 100% of the time. Image by Thomas Julou (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons.

Moving Targets

Even in controlled lab environments, gene drive has inherent limitations. The trait to be modified must be genetically determined—preferably by a single gene. Gene drive isn’t practical in organisms with long generation times, such as humans. And for the drive to spread, drive carriers must be able to infiltrate the natural population, ensuring that the drive is passed on to the normal organisms’ progeny. This means gene drive can’t affect organisms that don’t reproduce sexually.

These constraints aren’t a problem for all applications. There’s considerable excitement about eradicating mosquito-borne illnesses like malaria—a disease that kills hundreds of thousands of people every year and sickens hundreds of millions more—by using lethal gene drives to control mosquito species that transmit the disease, or even push them to extinction.

Although the goal is ambitious, tantalizing evidence suggests it might be possible. In 2015, researchers at the University of California in San Diego reported a proof-of-concept experiment on a caged population of flies, which showed that heterozygotes for a drive could pass it on to 95-100% of their progeny, as opposed to the Mendelian 50%.

But while it’s promising that an allele can be driven to high frequency using this method, there are reasons to suspect that the situation in the wild may be very different. “For this drive to work, it needs to recognize the site in the genome where it’s going to insert itself,” says Philipp Messer, a researcher at Cornell University studying the evolution of resistance to gene drives. “In those lab populations, they were all the same strain, so they always had exactly the same sequence at that target site. And that’s why it worked in all of those flies.” In wild genomes, there’s much more natural variation, so susceptibility and resistance will be less consistent.

According to Messer’s recent work, this standing variation might not even be the biggest source of resistance alleles. Resistance can also arise through other mechanisms, such as mutations caused by the drive mechanism itself. When DNA is cut, the cell doesn’t always fix the break using homology-directed repair. Instead, it can simply stitch the ends of the DNA together—a technique called non-homologous end joining. This often results in mutations, which could leave the target site unrecognizable to the driver system.

Messer’s group is conducting experiments to better understand how these resistance alleles arise. They release flies with a driver construct into a caged population of normal lab flies, and then closely monitor the flies to see when the drive comes close to fixation—that is, when nearly every individual to carry the gene drive at both loci. Once this happens, they check the population for the resistance alleles they predict will evolve.

According to Messer, one strategy to avoid such resistance is to use multiple guide sequences or to target an essential gene, but whether those techniques will work in practice hasn’t been tested. Otherwise, he says, the only way resistance alleles like this won’t emerge is if the population is so small that there’s essentially no variation at the target site and the rate of nonhomologous end joining can somehow be reduced to near zero. “People now would say, ‘Well, we’re really happy if we can have a 99% conversion rate,’” Messer says. A 99% conversion rate means nonhomologous end joining occurs in a seemingly trivial 1% of cases. But, Messer says, “If you have a population of a billion insects, that still means in every generation non-homologous end joining happens in ten million insects. You have a tremendous supply of these resistance alleles.”

Inbreeding Escape

It’s possible that these resistance mechanisms won’t take effect rapidly enough to prevent the drive from spreading through a population. By the time resistance crops up, we may already have achieved our goal. But even if resistance is slow to develop, gene drives face other hurdles in natural populations. Another evasion method is inbreeding, a topic studied by Professor James Bull at the University of Texas at Austin. In normal circumstances, inbreeding can harm a species by reducing genetic diversity, making it more likely to succumb to threats. But when harmful gene drives spread through a population, inbreeders may have an advantage. This edge arises because inbreeding increases the frequency of homozygotes in the population.

“If the population doesn’t have any heterozygotes at all, then individuals either don’t carry the gene drive or they’re homozygous for it—in which case, they’re dead,” Bull says about lethal gene drive. According to Bull, whether a species will evade gene drive via inbreeding depends on the magnitude of the genetic costs of inbreeding. “The offspring that are inbred aren’t quite as fit as the wild type,” Bull says, and in some species, the fitness of inbred offspring may be well below that of the wild type. But if this reduction in fitness is less detrimental than the drive, it’s very likely that inbreeding will sidestep the drive.

To circumvent these resistance mechanisms, some have proposed introducing multiple drives at once, but even this may not be enough to defeat nature. Cellular molecules that interfere with the Cas9 endonuclease protein itself or its expression could be enough to suppress multiple drives at once. “We’re all guessing right now. To me, that makes it somewhat exciting,” Bull says.

According to Michael Wade, a Distinguished Professor of Biology at Indiana University Bloomington, drive mechanisms might involve only a certain type of gene, but the genes that oppose them could be anywhere in the genome. But despite many means for populations to evolve resistance to gene drives, several successful drives exist in nature.

The Drosophila genome, for instance, harbors several copies of DNA sequences called P-elements, selfish genes that splice duplicates of themselves into random sites in the genome. Fifty years ago, P-elements didn’t even exist; now, you can’t find a fruit fly without them. But one key difference is that P-elements can insert themselves anywhere in the genome; they don’t require a precise target site like the CRISPR/Cas9 system does. And aside from sometimes inserting in an important gene, P-elements aren’t harmful to the flies, unlike the many proposed gene drives intended to disable or even exterminate their host species.

All-Natural Drives

Genes drives much less benign than P-elements have also spread in the wild. In natural fruit fly populations, a complex of driven genes called Segregation Distorter interferes with sperm production in males that possess one copy of the drive, causing dysfunction of spermatids that lack the drive. As a result, Segregation Distorter has spread through the natural population even though it diminishes carriers’ reproductive fitness.

Another strikingly selfish gene, called the t-allele, has also been found in some mouse populations. Males homozygous for the t-allele are sterile, but heterozygotes disproportionately produce sperm bearing the t-allele. The allele can thus be driven to high frequency despite causing serious fitness impairments in male carriers. Promisingly for gene drive hopefuls, this implies that it might also be possible to use CRISPR/Cas9 to force a deleterious selfish gene of our own design to high frequency.

By default, the t-allele could never become fixed in a population: all males would be sterile, and the population would die out. But nature also has examples of pushback against drive that are less easy to explain. According to Wade, the selfish gene Medea (found in flour beetles) defies our expectations: “In the simple mathematics of the Medea model, Medea spreads to fixation, yet we find populations in nature where Medea exists at an intermediate frequency.”

No one knows what genetic variants are responsible for hampering Medea’s spread, but something in nature must be stopping it from becoming fixed—at least at the predicted rate. The interplay between drives and resistance appears more complex than we understand.

Inhospitable Hosts

Medea isn’t an exceptional case. According to Wade, most drives in nature have suppressors. If a drive weakens the organism that carries it, there will be selection for resistance against the drive. That’s why some have argued that rather than using drives to reduce or eliminate the population that carries it, gene drive might better be used to tamper with a vector so that it can no longer support a pathogen—for instance, by altering a gene in mosquitoes that the malaria parasite requires to grow or spread.

A gene drive that has no negative effect on its mosquito host might be able to spread through a population without meeting resistance mechanisms in the insects. But Bull questions how much we actually know about what gene to alter. And even if we know which mosquito gene to target, that gene probably exists in the host for a reason. “Knocking out a gene may often have consequences,” Bull says. “It may not kill the individual if it lacks it, but if we’re knocking fitness down 10 or 20%, it’s still going to be a pretty strong selective advantage for the insect to evolve resistance.”

Instead of altering a vector gene needed by the parasite, some have suggested inserting whole new parasite-fighting genes into vectors’ genomes—and proof-of-concept experiments in lab populations have shown promising results. But in larger wild populations, the parasite’s response is unpredictable, and a mutation or two might allow it to grow again.

Even if the parasite can’t evolve resistance, selection may still favor flies without the drive, preventing it from establishing itself in the population. Driver alleles cut chromosomes, and off-target effects could disrupt important parts of the genome. Although this hasn’t seemed to stop P-elements in flies from spreading, it may still be a hindrance for other drives, and different organisms may not have as much tolerance for slicing up chromosomes.

Cheating Evolution

Beyond these molecular obstacles, gene drive may face difficulties at an even larger scale. For many applications, the drive must be able to spread from one local population to another—and predicting how likely that will be and how rapidly it might occur requires understanding how far individuals migrate, how fast they move, and other factors. And even if individuals of the targeted population readily intermingle with those of neighboring populations, the drive may face new challenges due to genetic variance in the neighbors.

The barriers don’t end here: there are almost certainly other problems we haven’t even foreseen, and many are likely species-specific. But maybe that’s not such a bad thing. “Let’s say you don’t want to eradicate the population, but you just want to severely weaken it,” says Messer. “You could drive a really deleterious allele to a population, it would spread there, the population would decline, and eventually only the few individuals with resistance alleles would survive.” The suppressed population could then be subjected to other challenges, such as pesticides. In the case of malaria-carrying mosquitos, their numbers might be reduced enough to halt the spread of malaria before the population rebounds.

Others are also hopeful that despite gene drive’s flaws, there’s reason to be optimistic. “I think having a new tool in the arsenal, gene drive, is great,” Wade comments. “I just don’t think that’s going to become the only tool.”

Although many call for caution and further study, the potential for massive benefits has others clamoring to release CRISPR/Cas9 gene drives into the wild. Whether or not we’re ready, our predictions about gene drive’s effectiveness may soon undergo field tests. Although resistance would be a disappointment to many, the fact that it seems so likely to develop could actually serve as a natural safeguard against the out-of-control drives that has prompted concern about the technology. “I think if we know one thing for certain, it will be difficult in practice to actually cheat evolution,” Messer says. After all, evolution has spent a lot longer practicing this game than we have.

More on gene drives at Genes to Genomes:
Gene drive: More research, not more regulations
Modeling the promise and peril of gene drive