Editor's Note: The second part of this article is “CRISPR Critters and the Fight Against Disease, Part 2: Gene drives give scientists the key to a Pandora’s box.”
First, let’s acknowledge the source of that irresistible pun in the headline. It’s lifted from a 2010 Lawrence Berkeley National Laboratory news release announcing the identification by a team led by University of California, Berkeley biochemist Jennifer Doudna of an enzyme — a so-called CRISPR-associated protein — that plays a key role in microbial immune systems.
Wait, microbes have immune systems? Who ever heard of a sick bacterium? What do they need immunity to?
Viruses, it turns out. Just like us human beings. Only different.
A little science
Every cell of every living thing on Earth is besieged and outnumbered, 10 to 1, by viruses. Having no machinery for replication of their own, viruses need to infiltrate and subvert the DNA or RNA of a host cell for their own propagation.
Multicelled organisms — mosquitoes, mice, deer, people — are bundles of eukaryotic cells whose outer membranes enclose a nucleus and internal, membrane-bound organelles. Eukaryotes have developed a complex immune system based on deploying specialized B and T cells that can learn to recognize dangerous invasive pathogens and swarm to destroy them or the cells infected by them.
Microbes, on the other hand — bacteria and archaea, single-celled organisms classified as prokaryotes — have no nuclei but protect themselves from potentially lethal viral hijacking by incorporating in their DNA “clustered regularly interspaced short palindromic repeats,” or CRISPR. That acronym was coined in 2002, 15 years after the phenomenon was observed by a Japanese researcher.
DNA is made up of four nucleic acid bases: adenine, cytosine, guanine and thymine, customarily abbreviated by the letters A, C, G and T. (Uracil, U, substitutes for thymine in RNA.) A genome can be thought of as consisting of a mindboggling litany of those letters — AACGTATTGCCAGGT and so on and on, about 3 billion times in a human — wound around one another in the two strands of a double helix. Some of the strings, parceled among 23 pairs of chromosomes, encode our 25,000 genes. Some perform other functions.
RPSIRC was I ere I saw CRISPR
A palindrome is a word or phrase that reads the same in either direction: noon, kayak, madam, was it a car or a cat I saw, or this variation on a famous quote apocryphally attributed to Napoleon. A snippet of DNA that reads GATACCATAG is, by analogy, palindromic.
In microbes, those palindromic CRISPR sequences range from 24 to 48 base pairs and are marked off by “spacers,” fragments of DNA from 30 to 60 nucleotides in length captured in the fight against an invading virus. They serve as a record of viral infections. More important, they provide a pattern for the transcription of CRISPR RNA and the activation of one or more of a set of 70 neighboring CRISPR-associated — or Cas — proteins, which express and refine the enzymes needed to neutralize the attacker. Since it’s a permanent modification of the genome, CRISPR-Cas immunity is passed along when the bacterium divides.
In 2012, Doudna, along with collaborators from Berkeley, the University of Vienna and Sweden’s Umeå University, published an even more groundbreaking discovery. If a particular CRISPR-associated protein, Cas9, was tagged with two engineered strands of “guide” RNA, it could read its way down a genome, latch onto a specific targeted sequence, cut both strands of DNA at that site, delete or amend the exposed sequence according to a template given it by the researchers, and stitch the corrected strands of the helix together again. In other words, nature has given scientists a ready-made tool for selectively editing an organism’s operating system.
Within a year, scientists including Doudna, Emmanuelle Charpentier at Umeå, Frank Church at Harvard and Feng Zhang at the Massachusetts Institute of Technology’s Broad Institute, had published papers describing applications of this simple, rapid and astonishingly accurate technique to modify the genomic machinery of eukaryotic cells. CRISPR became the hottest word in the genetic engineering vocabulary.
“In the following 8 months,” Science magazine reported in August 2013, “various groups have used it to delete, add, activate or suppress targeted genes in human cells, mice, rats, zebra fish, bacteria, fruit flies, yeast, nematodes and crops, demonstrating broad utility for the technique. With CRISPR, scientists can create mouse models of human diseases much more quickly than before, study individual genes much faster, and easily change multiple genes in cells at once to study their interactions.”
Both Berkeley and MIT’s Broad raced to file patents on the method — which were awarded initially to Zhang but are now in dispute. Meanwhile, in various combinations, all of the scientists have cooperated to found companies devoted to developing and commercializing CRISPR genome-editing technology.
Consider the mosquito
Now let’s shift gears. Let’s think about mosquitoes.
The earth is abuzz with mosquitoes. They and their larvae make up an appreciable percentage of the planet’s biomass. They’ve been around for more than 100 million years, breeding in standing water and, in the Arctic, swarming in clouds thick enough to suffocate a caribou. They’ve coevolved with myriad other life forms (fish, frogs, birds — avid consumers of mosquito-rich diets) into about 3,500 named species. Only a few hundred of these actually bite human beings. Nevertheless, more than 600 million people sicken each year worldwide from diseases transmitted by browsing mosquitoes, and at least 1 million die.
The toll on society is enormous, not just in terms of human suffering but also in lost productivity and expenditures for prevention and treatment. Malaria, caused by a parasitic protozoan that thrives in the gut of the female Anopheles mosquito and is passed along as it dips its proboscis into a buffet of human blood, infected 214 million people worldwide last year, according to the World Health Organization. More than 400,000 died. Malaria is a significant cause of poverty in the developing world, the WHO points out.
Up to 100 million cases of dengue fever, carried by the Aedes aegypti mosquito, are reported annually in 125 countries, the Centers for Disease Control and Prevention reports. It’s the fastest-growing vector-borne disease worldwide. A recent study of dengue in eight tropical countries estimated that the drag it exerts on their economies totals nearly $2 billion a year.
Closer to home, an outbreak of West Nile fever carried by the Culex pipiens mosquito cost just one California county — Sacramento — $2.28 million in 2005, the CDC calculated. Only 163 cases were reported, but the treatment of the most serious of them involved outpatient care at $6,300 per visit, inpatient care at $33,000 per hospitalization and extended care at $18,000 per nursing home stay.
A better way
International travel and a warming planet have brought previously unknown mosquito-borne viral threats (like West Nile and chikungunya) to U.S. shores. Ebola, with a terrifying fatality rate of 71 percent, has so far been confined to isolated cases of medical workers returning from Africa. Zika virus — which almost always involves only mild symptoms if any but can cause catastrophic birth defects if contracted by pregnant women — is carried by two native North American mosquito species, A. aegypti in southern regions and Aedes albopictus, also known as the Asian tiger mosquito, as far north as the Great Lakes. The CDC has recorded 2,517 known cases of Zika in the United States as of this writing (11,588 if territories like Puerto Rico are counted), 584 (1,396) affecting pregnant women. Of these only one has proved fatal, to an elderly man in Utah.
Scientists have been battling mosquitoes since the discovery of their role in transmitting malaria and yellow fever in the late 19th century. Prevention efforts — insecticide spraying indoors and out, sleeping under treated nets, covering exposed skin, using topical repellants, taking antimalarial drugs — have averted 6.2 million malaria deaths since 2001, the WHO estimates, a 60 percent reduction. Vaccines have virtually eliminated yellow fever throughout most of the Americas. Still, some 30,000 fatalities from the disease are recorded annually in Africa and tropical South America.
Efforts to create a malaria vaccine have not yet borne fruit. But molecular biologist Anthony James, at the University of California, Irvine, has taken another tack. He’s looking at a mosquito-borne disease prevention strategy focused on the vector rather than the victim. He’s breeding female Aedes mosquitoes that will resist the malaria and dengue parasites they’ve historically harbored. Their bites may itch, but they won’t infect.
James’ first task was to engineer mosquito genes for the production of antibodies to the protozoans that cause disease in humans, especially Plasmodium falsiparum, the most deadly parasite. Next, he confronted the daunting problem of how to get the gene out of the laboratory and into enough mosquitoes in the wild to pass the parasite-rejecting trait throughout the population.
Then he read the Doudna-Charpentier paper about CRISPR-Cas9.
The solution dawned.
More about that, about an even more exciting refinement called gene drive and about the future of the world’s mosquitoes (spoiler alert: perhaps not sunny) in the next installment: “CRISPR Critters and the Fight Against Disease, Part 2: Gene drives give scientists the key to a Pandora’s box.”
David Ollier Weber is a principal of The Kila Springs Group in Placerville, Calif., and is a regular contributor to H&HN Daily.