Crispr's Mysteries

CRISPR’S MYSTERIES While the world rallies rancisco Mojica was not the first to see repeats. “Don’t care about repeats so much,” CRISPR, but he was probably the first to he says that they would warn him. “There are round the gene- be smitten by it. He remembers the day many repeats in many organisms — we’ve Fin 1992 when he got his first glimpse of the known about them for years and still don’t editing tool that microbial immune system that would launch know how many of them work.” a biotechnology revolution. He was review- Today, much more is known about the is revolutionizing ing genome-sequence data from the salt- clustered, regularly interspaced short palin- biotechnology, basic loving microbe Haloferax mediterranei and dromic repeats that give CRISPR its name and noticed 14 unusual DNA sequences, each 30 help the CRISPR–Cas microbial immune sys- questions about how it bases long. They read roughly the same back- tem to destroy invading viruses. But although PANAUD PEHAU-ARNAUDET/JEAN-MARC wards and forwards, and they repeated every most in biomedicine have come to revere the PRANGISHVILI/GERARD QUAX/DAVID TESSA works and where it came 35 bases or so. Soon, he saw more of them. mechanics of the system — particularly of a from still loom large. Mojica was entranced, and made the repeats version called CRISPR–Cas9 — for the ways in a focus of his research at the University of which it can be harnessed to edit genes, Mojica Alicante in Spain. and other microbiologists are still puzzling over BY HEIDI LEDFORD It wasn’t a popular decision. His lab went some basic questions about the system and how years without funding. At meetings, Mojica it works. How did it evolve, and how did it shape would grab the biggest bigwigs he could find microbial evolution? Why do some microbes and ask what they thought of the strange little use it, whereas others don’t? And might it have

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Prokaryotes use CRISPR– other, yet-to-be- Maryland, and his colleagues have found1 a spacer becomes obsolete. And it can be a Cas to fight off viruses such appreciated roles a class of these mobile genetic elements that burden for microbes to retain extra DNA. “A as the one that formed this in their basic encodes the protein Cas1, which is involved bacterium cannot inflate its genome forever,” structure on a cell. biology? in inserting spacers into the genome. These says Rotem Sorek, a geneticist at the Weizmann “A lot of the ‘casposons’, he reasons, could have been the Institute of Science in Rehovot, Israel. attention paid to CRISPR systems in the media origin of CRISPR–Cas immunity. Researchers has really been around its use as a technology are now working to understand how these bits WHAT ELSE MIGHT IT BE DOING? — and with good reason. That’s where we’re of DNA hop from one place to another — and The origin of some spacers presents another seeing incredible impact and opportunities,” then to track how that mechanism may have mystery. Less than 3% of spacers observed so far says Jennifer Doudna, a molecular biologist at led to the sophistication of CRISPR–Cas. match any known sequences in DNA databases. the University of California, Berkeley, and one It could be a reflection of how little is known of the first scientists to reveal CRISPR–Cas’s HOW DOES IT WORK? about viruses. Most sequencing efforts have agility as a gene-editing tool. “At the same time, Many of the molecular details of how Cas pro- concentrated on those that infect people, live- there’s a lot of interesting fundamental biology teins add spacers have been worked out in fine stock or crops. “We know very little about the research to be done.” detail2 in recent years. But viral DNA is chemi- enemies of bacteria, and especially the enemies cally nearly identical to host DNA. How, in a cell of crazy archaea,” says Michael Terns, an RNA WHERE DID IT COME FROM? packed with DNA, do the proteins know which biologist at the University of Georgia in Athens. The biological advantages of something like DNA to add to the CRISPR–Cas memory? It is also possible that some spacers are the CRISPR–Cas are clear. Prokaryotes — bacteria The stakes are high: if a bacterium adds a ghosts of viruses no longer around or mutated and less-well-known single-celled organisms piece of its own DNA, it risks suicide by auto- beyond recognition. But a third possibility has called archaea, many of which live in extreme immune attack, says Virginijus Siksnys, a the field buzzing. Researchers have found exam- environments — face a constant onslaught of genetic invaders. Viruses outnumber prokaryotes by ten to one and are said to kill half of the world’s bacteria every two days. Prokary- “A MAJOR QUESTION IS HOW MUCH BIOLOGY otes also swap scraps of DNA called plasmids, which can be parasitic — draining resources from their host and forcing it to self-destruct IS THERE THAT GOES BEYOND DEFENCE.” if it tries to expel its molecular hitch-hiker. It seems as if nowhere is safe: from soil to sea to the most inhospitable places on the planet, biochemist at Vilnius University in Lithuania. ples of CRISPR–Cas systems doing more than genetic invaders are present. “These enzymes are a double-edged sword.” warding off genetic intruders. In some bacteria, Prokaryotes have evolved a slew of weapons It may be that populations of bacteria and CRISPR–Cas components control DNA repair, to cope with these threats. Restriction enzymes, archaea can absorb some error, says Rodolphe gene expression and the formation of biofilms. for example, are proteins that cut DNA at or near Barrangou, a microbiologist at North Carolina They can also determine a bacterium’s ability a specific sequence. But these defences are blunt. State University in Raleigh. A few cellular sui- to infect others: Legionella pneumophila, which Each enzyme is programmed to recognize cer- cides may not matter if other cells can thrive causes Legionnaires’ disease, must have the tain sequences, and a microbe is protected only after a viral attack. Cas protein Cas2 in order to infect the amoeba if it has a copy of the right gene. CRISPR–Cas In fact, when viruses infiltrate a bacterial that is its natural host. “A major question is is more dynamic. It adapts to and remembers ecosystem, often only about one bacterium in how much biology is there that goes beyond specific genetic invaders in a similar way to how 10 million will gain a spacer that lets it defend defence,” says Erik Sontheimer, a molecular human antibodies provide long-term immunity itself. Those odds make it hard to study what biologist at the University of Massachusetts after an infection. “When we first heard about drives spacer acquisition, and to learn why a cell Medical School in Worcester. “That is some- this hypothesis, we thought that would be way succeeded where others failed. “It’s difficult to thing where there’s still quite a few shoes to drop too sophisticated for simple prokaryotes,” says catch that bacterium when it actually is happen- in the coming years.” microbiologist John van der Oost of Wagenin- ing,” says Luciano Marraffini, a microbiologist Sontheimer adds that it creates an enticing gen University in the Netherlands. at the Rockefeller University in New York City. parallel with the discovery of RNA interfer- Mojica and others deduced the function of Sorting out how suitable spacers are recog- ence, a system that silences gene expression CRISPR–Cas when they saw that DNA in the nized — and boosting the rate at which they in plants, animals and other non-prokaryotic spaces between CRISPR’s palindromic repeats are incorporated — could be useful. Some organisms. RNA interference was also primar- sometimes matches sequences in viral genomes. work has shown that cells containing CRISPR– ily thought of as a defence mechanism early on, Since then, researchers have worked out that Cas machinery could serve as a recording and it was only later that researchers noticed its certain CRISPR-associated (Cas) proteins add device of sorts, cataloguing DNA and RNA role in regulating host gene expression. these spacer sequences to the genome after bac- sequences that they have encountered3. This This could also explain why some spacers do teria and archaea are exposed to specific viruses might allow researchers to track a cell’s gene not match known viruses or plasmids, says Stan or plasmids. RNA made from those spacers expression or exposure to environmental Brouns, a microbiologist at Delft University of directs other Cas proteins to chew up any invad- chemicals over time. Technology in the Netherlands. “The systems ing DNA or RNA that matches the sequence. Researchers would also like to learn how are not tuned to be perfect: they grab the viral How did bacteria and archaea come to pos- old memories are pruned from the collec- DNA as well as their own,” he says. “As soon as sess such sophisticated immune systems? That tion. Most microbes with CRISPR–Cas sys- they start pulling in new pieces of DNA, they question has yet to be answered, but the lead- tems contain a few dozen spacers; some have can gain new functions — if they don’t die.” ing theory is that the systems are derived from only one. The archaeon Sulfolobus tokodaii, transposons — ‘jumping genes’ that can hop by contrast, dedicates 1% of its genome to its WHY DO ONLY SOME MICROBES USE IT? from one position to another in the genome. 5 CRISPR–Cas systems, including 458 spacers. Whatever other functions CRISPR–Cas has, Evolutionary biologist Eugene Koonin of the There may be little incentive to hang on to old it is clear that some microbes use it more US National Institutes of Health in Bethesda, spacers: if a virus mutates to avoid CRISPR–Cas, than others. More than 90% of archaea have

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LASTING PROTECTION don’t play favourites. Instead, they tend to mix and match different systems, quickly picking About 90% of known archaea and one-third of bacteria have some form of CRISPR–Cas immunity. NATURE This is controlled by a cluster of short DNA repeats separated by ‘spacer’ sequences and a series up new ones from other bacteria and discard- of nearby genes that encode CRISPR-associated (Cas) proteins. ing the old. Researchers have officially recognized 6 dif- IMMUNIZATION IMMUNITY ferent types of CRISPR system, with 19 sub- NIK SPENCER/ types. “And we really only know how a fraction Virus Bacterial of them actually work,” says Marraffini. cell wall Unravelling those mechanisms could hold the key to finding new biotechnological appli- cations for CRISPR–Cas systems. The beloved CRISPR–Cas9, for example, is a type II system, RNA transcribed which uses RNA molecules transcribed from Spacer from the spacers spacer sequences to direct an enzyme to cut sequence guides Cas proteins invading viral or plasmid DNA (see ‘Lasting to invading DNA or RNA so that it can protection’). But enzymes in type VI systems 6 Guide RNA be disarmed. — discovered last year — cut up RNA rather than DNA. And type IV systems contain Spacers are sequences captured from invading some genes associated with CRISPR–Cas, but viruses, which are Cas cleavage lack the repeats and the machinery to insert incorporated into the Repeat Repeat complex spacers. bacterial genome. Spacer Type III systems are among the most com- monly found CRISPR–Cas systems in nature — and among the least understood. Evidence Bacterial 1 2 3 4 chromosome so far suggests that they respond not to the cas genes invading DNA or RNA itself, but to the process Spacers of transcribing DNA into RNA. If that proves CRISPR array to be the case, it would be a new form of regula- tion that could expand the CRISPR–Cas tool- CRISPR-based immunity, whereas only about only a few types of virus to contend with4,5. box for genome editing, says Doudna. one-third of sequenced bacteria bother with it, CRISPR–Cas spacers can record a limited Other systems may yet crop up, particularly says Koonin. And no non-prokaryotic organ- number of viral sequences before the added as researchers extend their search beyond isms, even single-celled ones, have been caught DNA becomes a genomic burden. If the microbes that have been grown in culture, to troubling with CRISPR–Cas at all. diversity of viruses in the environment greatly include genetic sequences from environmental One archaeon, called Nanoarchaeum outweighs the number of possible spacers, DNA samples. “We have already said a cou- equitans, lives as a parasite on another CRISPR–Cas systems may be of little use, says ple of times that we reached the end,” says van archaeon in near-boiling waters and has Koonin. Another possibility is that archaea in der Oost — only to be surprised when a new dispensed with many of its genes related to extreme environments cannot rely as heavily CRISPR–Cas system surfaced. energy production and general cellular house- on other means of defence. One common way For Mojica, exploring that diversity and keeping. Yet in its minuscule, 490,000-letter answering basic questions about CRISPR DNA instruction manual, N. equitans has systems hold more allure than the revolution held on to a CRISPR–Cas system with about they sparked. This puzzles many of his col- 30 spacers. “A big chunk of its genome is still “CRISPR MUST BE SO leagues, he says. He has immersed himself in dedicated to CRISPR,” says Malcolm White, CRISPR–Cas biology for a quarter of a century, a molecular biologist at the University of and although there’s a lot of funding available St Andrews, UK. “CRISPR must be so impor- IMPORTANT, YET for those who wish to edit genomes, there is tant, yet we don’t really know why.” considerably less for the kind of work he does. Such differences suggest that there are key “I know that it’s a great tool. It’s fantastic. It ecological factors that favour CRISPR–Cas WE DON’T REALLY could be used to cure diseases,” says Mojica. systems, prizing viral defence — or other “But it’s not my business. I want to know how benefits — over the risks of cellular suicide, the system works from the very beginning to says Edze Westra, a microbiologist at the KNOW WHY.” the end.” ■ Penryn campus of the University of Exeter, UK. Extreme environments seem to favour for bacteria to thwart invaders is to mutate Heidi Ledford is a senior reporter for Nature CRISPR–Cas systems, but Westra notes that the proteins found in their own outer casing, in London. the frequency of such systems also varies called an envelope. Some archaea, however, 1. Krupovic, M., Makarova, K. S., Forterre, P., among bacteria in more-hospitable habitats. may have less freedom to tinker with these Prangishvili, D. & Koonin, E. V. BMC Biol. 12, 36 The bird pathogen Mycoplasma gallisepticum, envelopes because the envelopes’ structure is (2014). for example, tossed out its CRISPR–Cas equip- so crucial to the organism’s survival in harsh 2. Nuñez, J. K., Lee, A. S. Y., Engelman, A. & ment when it switched hosts from chickens to conditions. “This makes alternative systems Doudna, J. A. Nature 519, 193–198 (2015). 3. Shipman, S. L., Nivala, J., Macklis, J. D. & wild finches. Why the system was useful in a such as CRISPR more relevant,” says Mojica. Church, G. M. Science 353, aaf1175 (2016). chicken but not a finch is anyone’s guess, says 4. Weinberger, A. D., Wolf, Y. I., Lobkovsky, A. E., Westra. HOW MANY FLAVOURS OF CRISPR–CAS EXIST? Gilmore, M. S. & Koonin, E. V. mBio 3, e00456-12 (2012). Mathematical models and some early labo- Humans tend to focus on the CRISPR–Cas9 5. Westra, E. R. et al. Curr. Biol. 25, 1043–1049 (2015). ratory experiments suggest that CRISPR–Cas system, which is prized for its simplicity and 6. Abudayyeh, O. O. et al. Science 353, aaf5573 may be more of an advantage when there are versatility in genome editing, but microbes (2016).

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CORRECTION An earlier version of the graphic in this article misrepresented the nuclease activity of the Cas cleavage complex. It has been corrected.