Three years ago, scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells. Since then, the method has taken the scientific community by storm, with thousands of labs using it for applications from biomedicine to agriculture. Yet, the preceding 20-year journey—the discovery of a strange microbial repeat sequence; its recognition as an adaptive immune system; its biological characterization; and its repurposing for genome engineering—remains little known. This Perspective aims to fill in this backstory—the history of ideas and the stories of pioneers—and draw lessons about the remarkable ecosystem underlying scientific discovery.

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Introduction It’s hard to recall a revolution that has swept biology more swiftly than CRISPR. Just 3 years ago, scientists reported that the CRISPR system—an adaptive immune system used by microbes to defend themselves against invading viruses by recording and targeting their DNA sequences—could be repurposed into a simple and reliable technique for editing, in living cells, the genomes of mammals and other organisms. CRISPR was soon adapted for a vast range of applications—creating complex animal models of human-inherited diseases and cancers; performing genome-wide screens in human cells to pinpoint the genes underlying biological processes; turning specific genes on or off; and genetically modifying plants—and is being used in thousands of labs worldwide. The prospect that CRISPR might be used to modify the human germline has stimulated international debate. Medawar, 1968 Medawar P. Lucky Jim. If there are molecular biologists left who have not heard of CRISPR, I have not met them. Yet, if you ask scientists how this revolution came to pass, they often have no idea. The immunologist Sir Peter Medawar observed, “The history of science bores most scientists stiff” (). Indeed, scientists focus relentlessly on the future. Once a fact is firmly established, the circuitous path that led to its discovery is seen as a distraction. Yet, the human stories behind scientific advances can teach us a lot about the miraculous ecosystem that drives biomedical progress—about the roles of serendipity and planning, of pure curiosity and practical application, of hypothesis-free and hypothesis-driven science, of individuals and teams, and of fresh perspectives and deep expertise. Such understanding is important for government agencies and foundations that together invest, in the U.S. alone, more than $40 billion in biomedical research. It is also important for a general public who often imagines scientists as lone geniuses cloistered in laboratories. And, for trainees, it is especially valuable to have a realistic picture of scientific careers, as both guide and inspiration. Figure 1 Class 2, Type II CRISPR-Cas9 System from Streptococcus thermophilus Show full caption Type II systems are the simplest of the three types of CRISPR systems and have been the basis for genome editing technology. (A) The locus contains a CRISPR array, four protein-coding genes (cas9, cas1, cas2, and cns2) and the tracrRNA. The CRISPR array contains repeat regions (black diamonds) separated by spacer regions (colored rectangles) derived from phage and other invading genetic elements. The cas9 gene encodes a nuclease that confers immunity by cutting invading DNA that matches existing spacers, while the cas1, cas2, and cns2 genes encode proteins that function in the acquisition of new spacers from invading DNA. (B) The CRISPR array and the tracrRNA are transcribed, giving rise to a long pre-crRNA and a tracrRNA. (C) These two RNAs hybridize via complementary sequences and are processed to shorter forms by Cas9 and RNase III. (D) The resulting complex (Cas9 + tracrRNA + crRNA) then begins searching for the DNA sequences that match the spacer sequence (shown in red). Binding to the target site also requires the presence of the protospacer adjacent motif (PAM), which functions as a molecular handle for Cas9 to grab on to. (E) Once Cas9 binds to a target site with a match between the crRNA and the target DNA, it cleaves the DNA three bases upstream of the PAM site. Cas9 contains two endonuclease domains, HNH and RuvC, which cleave, respectively, the complementary and non-complementary strands of the target DNA, creating blunt ends. Over the past several months, I have sought to understand the 20-year backstory behind CRISPR, including the history of ideas and the stories of individuals. This Perspective is based on published papers, personal interviews, and other materials—including rejection letters from journals. At the end, I try to distill some general lessons. (As background, Figure 1 provides a brief overview of a type II CRISPR system, the variety that has been repurposed for genome engineering.) Most of all, the Perspective describes an inspiring ensemble of a dozen or so scientists who—with their collaborators and other contributors whose stories are not elaborated here—discovered the CRISPR system, unraveled its molecular mechanisms, and repurposed it as a powerful tool for biological research and biomedicine. Together, they are the Heroes of CRISPR.

CRISPR Is an Adaptive Immune System During the August holiday in 2003, Mojica escaped the scorching heat of Santa Pola’s beaches and took refuge in his air-conditioned office in Alicante. By now the clear leader in the nascent CRISPR field, he had turned his focus from the repeats themselves to the spacers that separated them. Using his word processor, Mojica painstakingly extracted each spacer and inserted it into the BLAST program to search for similarity with any other known DNA sequence. He had tried this exercise before without success, but the DNA sequence databases were continually expanding and this time he struck gold. In a CRISPR locus that he had recently sequenced from an E. coli strain, one of the spacers matched the sequence of a P1 phage that infected many E. coli strains. However, the particular strain carrying the spacer was known to be resistant to P1 infection. By the end of the week, he had slogged through 4,500 spacers. Of 88 spacers with similarity to known sequences, two-thirds matched viruses or conjugative plasmids related to the microbe carrying the spacer. Mojica realized that CRISPR loci must encode the instructions for an adaptive immune system that protected microbes against specific infections. Mojica et al., 2005 Mojica F.J.M.

Díez-Villaseñor C.

García-Martínez J.

Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Mojica went out to celebrate with colleagues over cognac and returned the next morning to draft a paper. So began an 18-month odyssey of frustration. Recognizing the importance of the discovery, Mojica sent the paper to Nature. In November 2003, the journal rejected the paper without seeking external review; inexplicably, the editor claimed the key idea was already known. In January 2004, the Proceedings of the National Academy of Sciences decided that the paper lacked sufficient “novelty and importance” to justify sending it out to review. Molecular Microbiology and Nucleic Acid Research rejected the paper in turn. By now desperate and afraid of being scooped, Mojica sent the paper to Journal of Molecular Evolution. After 12 more months of review and revision, the paper reporting CRISPR’s likely function finally appeared on February 1, 2005 (). At about the same time, CRISPR was the focus of attention in another, rather unlikely, venue: a unit of the French Ministry of Defense, some 30 miles south of Paris. Gilles Vergnaud, a human geneticist trained at the Institut Pasteur, had received doctoral and post-doctoral support from the Direction Générale de l’Armement. When he completed his studies in 1987, he joined the government agency to set up its first molecular biology lab. For the next 10 years, Vergnaud continued his work on human genetics. But when intelligence reports in the late 1990s raised concerns that Saddam Hussein’s regime in Iraq was developing biological weapons, the Ministry of Defense asked Vergnaud in 1997 to shift his group’s focus to forensic microbiology—developing methods to trace the source of pathogens based on subtle genetic differences among strains. Establishing a joint lab with the nearby Institute of Genetics and Microbiology at Université Paris-Sud, he set out to use tandem-repeat polymorphisms—which were the workhorse of forensic DNA fingerprinting in humans—to characterize strains of the bacteria responsible for anthrax and plague. Pourcel et al., 2005 Pourcel C.

Salvignol G.

Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. The French Defense Ministry had access to a unique trove of 61 Y. pestis samples from a plague outbreak in Vietnam in 1964–1966. Vergnaud found that these closely related isolates were identical at their tandem-repeat loci—with a sole exception of a site that his colleague Christine Pourcel discovered was the CRISPR locus. The strains occasionally differed by the presence of new spacers, which were invariably acquired in a polarized fashion at the “front” end of the CRISPR locus (). Strikingly, many of the new spacers corresponded to a prophage present in the Y. pestis genome. The authors proposed that the CRISPR locus serves in a defense mechanism—as they put it, poetically, “CRISPRs may represent a memory of ‘past genetic aggressions.’” Vergnaud’s efforts to publish their findings met the same resistance as Mojica’s. The paper was rejected from the Proceedings of the National Academy of Sciences, Journal of Bacteriology, Nucleic Acids Research, and Genome Research, before being published in Microbiology on March 1, 2005. Bolotin et al., 2005 Bolotin A.

Quinquis B.

Sorokin A.

Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Finally, a third researcher—Alexander Bolotin, a Russian émigré who was a microbiologist at the French National Institute for Agricultural Research—also published a paper describing the extrachromosomal origin of CRISPR, in Microbiology in September 2005 (). His report was actually submitted a month after Mojica’s February 2005 paper had already appeared—because his submission to another journal had been rejected. Notably, Bolotin was the first to speculate how CRISPR conferred immunity—proposing that transcripts from the CRISPR locus worked by anti-sense RNA inhibition of phage gene expression. Although reasonable, the guess would prove to be wrong.

Experimental Evidence that CRISPR Confers Adaptive Immunity and Employs a Nuclease Like Mojica, Philippe Horvath could hardly have chosen a thesis topic that was more local or less sexy. As a Ph.D. student at the University of Strasbourg, he concentrated on the genetics of a lactic-acid bacteria used in the production of sauerkraut—the central ingredient in the Alsatian specialty choucroute garnie. Given his interest in food science, Horvath skipped doing post-doctoral research and in late 2000 joined Rhodia Food, a maker of bacterial starter cultures located in Dangé-Saint-Romain in western France, to set up its first molecular biology lab. The company was later acquired by the Danish firm Danisco, which was itself acquired by DuPont in 2011. Rhodia Food was interested in Horvath’s microbiological skills because other lactic-acid bacteria, such as Streptococcus thermophilus, are used to make dairy products, such as yogurt and cheese. Horvath’s mission included developing DNA-based methods for precise identification of bacterial strains and overcoming the frequent phage infections that plagued industrial cultures used in dairy fermentation. Understanding how certain S. thermophilus strains protect themselves from phage attack was thus of both scientific interest and economic importance. After learning about CRISPR at a Dutch conference on lactic-acid bacteria in late 2002, Horvath began using it to genotype his strains. By late 2004, he noticed a clear correlation between spacers and phage resistance—as would be reported just a few months later by Mojica and Vergnaud. In 2005, Horvath and colleagues—including Rodolphe Barrangou, a newly minted Ph.D. at Danisco USA, and Sylvain Moineau, a distinguished phage biologist at Université Laval in Québec City—set out to directly test the hypothesis that CRISPR was an adaptive immune system. Notably, Moineau had also been an industrial scientist. He had earned his Ph.D in Food Sciences at Laval, also studying lactic-acid bacteria, and had worked at Unilever Corporation before returning to academia at Laval; he had been collaborating with Rhodia Food since 2000. Barrangou et al., 2007 Barrangou R.

Fremaux C.

Deveau H.

Richards M.

Boyaval P.

Moineau S.

Romero D.A.

Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Using a well-characterized phage-sensitive S. thermophilus strain and two bacteriophages, these investigators performed genetic selections to isolate phage-resistant bacteria. Rather than harboring classical resistance mutations (such as in a cell-surface receptor required for phage entry), the resistant strains had acquired phage-derived sequences at their CRISPR loci (). Moreover, the insertion of multiple spacers correlated with increased resistance. They had seen acquired immunity in action. Bolotin et al., 2005 Bolotin A.

Quinquis B.

Sorokin A.

Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Makarova et al., 2006 Makarova K.S.

Grishin N.V.

Shabalina S.A.

Wolf Y.I.

Koonin E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. They also studied the role of two of the cas genes: cas7 and cas9. Bacteria required cas7 in order to gain resistance, but those carrying a phage-derived spacer did not need the gene to remain resistant—suggesting that Cas7 was involved in generating new spacers and repeats, but not in immunity itself. In contrast, cas9—whose sequence contained two types of nuclease motifs (HNH and RuvC) and whose product thus presumably cut nucleic acids ()—was necessary for phage resistance; the Cas9 protein was an active component of the bacterial immune system. (Warning: In the early CRISPR literature, the now-famous cas9 gene was called cas5 or csn1.) Finally, they found that rare phage isolates that overcame CRISPR-based immunity carried single-base changes in their genomes that altered the sequence corresponding to the spacers. Immunity thus depended on a precise DNA sequence match between spacer and target.

Programming CRISPR Makarova et al., 2006 Makarova K.S.

Grishin N.V.

Shabalina S.A.

Wolf Y.I.

Koonin E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. John van der Oost, who got his Ph.D. from the Free University of Amsterdam in 1989, originally set out to solve the world’s clean-energy needs by using cyanobacteria to produce biofuels. He studied metabolic pathways in bacteria, working in Helsinki and Heidelberg before returning to Amsterdam. In 1995, Wageningen University offered him a permanent position—but with a catch: they wanted him to expand a group working on extremophile microbes. van der Oost, who had once heard a talk while in Germany about Sulfolobus solfataricus, which thrives in the hot springs of Yellowstone National Park, was game to investigate the evolutionary differences in the metabolic pathways of these strange microbes. He began to collaborate with Eugene Koonin—an expert in microbial evolution and computational biology at the National Center for Biotechnology Information (NCBI) at the National Institutes of Health. Koonin had begun working on classifying and analyzing CRISPR systems, and on a visit in 2005, he introduced van der Oost to the then-obscure field of CRISPR (). van der Oost had just received a major grant from the Dutch National Science Foundation. In addition to working on the problem described in his proposal, he decided to use some of the funding to study CRISPR. (In his report to the agency 5 years later, he underscored the value of the agency’s policy of allowing researchers the freedom to shift their scientific plans.) Brouns et al., 2008 Brouns S.J.J.

Jore M.M.

Lundgren M.

Westra E.R.

Slijkhuis R.J.H.

Snijders A.P.L.

Dickman M.J.

Makarova K.S.

Koonin E.V.

van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. He and his colleagues inserted an E. coli CRISPR system into another E. coli strain that lacked its own endogenous system. This allowed them to biochemically characterize a complex of five Cas proteins, termed Cascade (). (E. coli has the more complex Class 1, type I CRISPR system, in which the functions of Cas9 are instead performed by the Cascade complex, together with the nuclease Cas3. See Table 1 .) Sorek et al., 2008 Sorek R.

Kunin V.

Hugenholtz P. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. By knocking out each component individually, they showed that Cascade is required for cleaving a long precursor RNA, transcribed from the CRISPR locus, into 61-nucleotide-long CRISPR RNAs (crRNAs). They cloned and sequenced a set of crRNAs that co-purified with the Cascade complex and found that all started with the last eight bases of the repeat sequence, followed by the complete spacer and the beginning of the next repeat region. This finding supported earlier suggestions that the palindromic nature of the repeats would lead to secondary structure formation in the crRNA (). To prove that the crRNA sequences are responsible for CRISPR-based resistance, they set out to create the first artificial CRISPR arrays—programming CRISPR to target four essential genes in lambda (λ) phage. As they predicted, the strains carrying the new CRISPR sequence showed resistance to phage λ. It was the first case of directly programming CRISPR-based immunity—a flu shot for bacteria. The results also hinted that the target of CRISPR was not RNA (as Bolotin had proposed) but, rather, DNA. The authors had designed two versions of the CRISPR array—one in the anti-sense direction (complementary to both the mRNA and coding strand of the DNA locus) and one in the sense direction (complementary only to the other DNA strand). Although the spacers varied in their efficacy, the fact that the sense version worked strongly suggested that the target was not mRNA. Still, the evidence was indirect. With the journal editors urging caution about drawing a firm conclusion, van der Oost’s paper in Science offered the notion that CRISPR targets DNA as a “hypothesis.”

CRISPR Targets DNA Luciano Marraffini was finishing his Ph.D., working on Staphylococcus, at the University of Chicago, when he learned about CRISPR from Malcolm Casadaban, a faculty member in the department who was a world authority on phage genetics. Casadaban had immediately seen the importance of discovery in 2005 that CRISPR was likely to be an adaptive immune system and talked about CRISPR to everyone who would listen. Like many in the phage community, Marraffini was convinced that CRISPR could not work by RNA interference because this mechanism would be too inefficient to overcome the explosive growth that occurs upon phage infection. Instead, he reasoned, CRISPR must cut DNA—functioning, in effect, like a restriction enzyme. Marraffini was eager to pursue post-doctoral work in one of the handful of groups in the world studying CRISPR, but his wife had a good job as a translator in the Cook County, Illinois criminal courts and he felt he should stay in Chicago. He persuaded Erik Sontheimer, a biochemist at Northwestern University who had been working on RNA splicing and RNA interference, to let him join his lab to work on CRISPR. Marraffini and Sontheimer, 2008 Marraffini L.A.

Sontheimer E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Even before moving to Northwestern, Marraffini started working on CRISPR even as he completed his graduate work—exploring whether the Staphylococcus CRISPR system could block plasmid conjugation. He noticed that a strain of Staphylococcus epidermidis had a spacer that matches a region of the nickase (nes) gene encoded on plasmids from antibiotic-resistant Staphylococcus aureus. He showed that these plasmids cannot be transferred to S. epidermidis but that disrupting either the nes sequence in the plasmid or the matching spacer sequences in the CRISPR locus in the genome abolishes interference (). Clearly, CRISPR blocked the plasmids, just as it blocked viruses. Marraffini and Sontheimer thought briefly about trying to reconstitute the CRISPR system in vitro to demonstrate that it cuts DNA. But the S. epidermidis system was too complicated—it had nine cas genes—and it was still too poorly characterized. Instead, they turned to molecular biology. Cleverly, they modified the nes gene in the plasmid targeted by the CRISPR system—inserting a self-splicing intron in the middle of its sequence. If CRISPR targeted mRNA, the change would not affect interference because the intronic sequence would be spliced out. If CRISPR targeted DNA, the insertion would abolish interference because the spacer would no longer match. The results were clear: the target of CRISPR was DNA. Sontheimer and Marraffini, 2008 Sontheimer, E., and Marraffini, L. (2008). Target DNA interference with crRNA. U.S. Provisional Patent Application 61/009,317, filed September 23, 2008; later published as US2010/0076057 (abandoned). Marraffini and Sontheimer recognized that CRISPR was essentially a programmable restriction enzyme. Their paper was the first to explicitly predict that CRISPR might be repurposed for genome editing in heterologous systems. “From a practical standpoint,” they declared, “the ability to direct the specific addressable destruction of DNA that contains any given 24- to 48-nucleotide target sequence could have considerable functional utility, especially if the system can function outside of its native bacterial or archaeal context.” They even filed a patent application including the use of CRISPR to cut or correct genomic loci in eukaryotic cells, but it lacked sufficient experimental demonstration and they eventually abandoned it ().

Cas9 Is Guided by crRNAs and Creates Double-Stranded Breaks in DNA Barrangou et al., 2007 Barrangou R.

Fremaux C.

Deveau H.

Richards M.

Boyaval P.

Moineau S.

Romero D.A.

Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Following the seminal study in 2007 confirming that CRISPR is an adaptive immune system (), Sylvain Moineau continued to collaborate with Danisco to understand the mechanism by which CRISPR cleaves DNA. Garneau et al., 2010 Garneau J.E.

Dupuis M.-È.

Villion M.

Romero D.A.

Barrangou R.

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Horvath P.

Magadán A.H.

Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. The problem was that CRISPR was normally so efficient that Moineau and his colleagues could not readily observe how invading DNA was destroyed. However, they caught a lucky break while studying plasmid interference in S. thermophilus. The investigators found a handful of bacterial strains in which CRISPR conferred only partial protection against plasmid transformation by electroporation. In one such inefficient strain, they could see linearized plasmids persisting inside the cells. Somehow, the process of plasmid interference had been slowed down enough to observe the direct products of CRISPR’s action (). Barrangou et al., 2007 Barrangou R.

Fremaux C.

Deveau H.

Richards M.

Boyaval P.

Moineau S.

Romero D.A.

Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Deveau et al., 2008 Deveau H.

Barrangou R.

Garneau J.E.

Labonté J.

Fremaux C.

Boyaval P.

Romero D.A.

Horvath P.

Moineau S. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Horvath et al., 2008 Horvath P.

Romero D.A.

Coûté-Monvoisin A.-C.

Richards M.

Deveau H.

Moineau S.

Boyaval P.

Fremaux C.

Barrangou R. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. This strain allowed them to dissect the process of cutting. Consistent with their earlier results (), they showed that the cutting of the plasmid depended on the Cas9 nuclease. When they sequenced the linearized plasmids, they found a single precise blunt-end cleavage event 3 nucleotides upstream of the proto-spacer adjacent motif (PAM) sequence, a key sequence feature whose function they had characterized in earlier papers (). Expanding their analysis, they showed that viral DNA is also cut in precisely the same position relative to the PAM sequence. Moreover, the number of distinct spacers matching a target corresponded to the number of cuts observed. Their results showed definitively that Cas9’s nuclease activity cut DNA at precise positions encoded by the specific sequence of the crRNAs.

Discovery of tracrRNA Despite intense study of the CRISPR-Cas9 system, one additional piece of the puzzle was missing—a small RNA that would come to be called trans-activating CRISPR RNA (tracrRNA). In fact, the discoverers, Emmanuelle Charpentier and Jörg Vogel, were not specifically looking to study the CRISPR system; they were simply trying to identify microbial RNAs. Mangold et al., 2004 Mangold M.

Siller M.

Roppenser B.

Vlaminckx B.J.M.

Penfound T.A.

Klein R.

Novak R.

Novick R.P.

Charpentier E. Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule. Charpentier had earned her Ph.D. in microbiology from Pasteur Institute in 1995 and did post-doctoral work in New York for 6 years before starting her own lab at the University of Vienna in 2002 and Umeå, Sweden in 2008. After discovering an unusual RNA that controls virulence in Streptococcus pyogenes (), she became interested in identifying additional regulatory RNAs in microbes. She used bioinformatics programs to scan intergenic regions in S. pyogenes for structures, suggesting that they might encode non-coding RNAs. She had found several candidate regions—including one near the CRISPR locus—but they were hard to follow up without direct information about the RNAs themselves. Sharma et al., 2010 Sharma C.M.

Hoffmann S.

Darfeuille F.

Reignier J.

Findeiss S.

Sittka A.

Chabas S.

Reiche K.

Hackermüller J.

Reinhardt R.

et al. The primary transcriptome of the major human pathogen Helicobacter pylori. The solution appeared when Charpentier met Vogel at the 2007 meeting of RNA Society in Madison, Wisconsin. Trained as a microbiologist in Germany, Vogel had begun focusing on finding RNAs in pathogens during his postdoctoral work in Uppsala and Jerusalem and had continued this work when he started his own group in 2004 at the Max Planck Institute for Infection Biology in Berlin. (Five years later, he would move to Würzburg to lead a research center on infectious disease.) With the recent advent of “next-generation sequencing” technology, Vogel realized that massively parallel sequencing would make it possible to produce comprehensive catalogs of any microbial transcriptome. He had just applied the approach to Helicobacter pylori, the bacterium responsible for stomach ulcers (), and was working on various other bugs. Charpentier and Vogel decided to turn the shotgun on S. pyogenes as well. Deltcheva et al., 2011 Deltcheva E.

Chylinski K.

Sharma C.M.

Gonzales K.

Chao Y.

Pirzada Z.A.

Eckert M.R.

Vogel J.

Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. The approach yielded a striking result: the third-most abundant class of transcript—after only ribosomal RNA and transfer RNA—was a novel small RNA that was transcribed from a sequence immediately adjacent to the CRISPR locus (in the region that had caught Charpentier’s attention) and had 25 bases of near-perfect complementary to the CRISPR repeats. The complementarity suggested that this tracrRNA and the precursor of the crRNAs hybridized together and were processed into mature products by RNaseIII cleavage. Genetic deletion experiments confirmed this notion, showing that tracrRNA was essential for processing crRNAs and thus for CRISPR function (). Jinek et al., 2012 Jinek M.

Chylinski K.

Fonfara I.

Hauer M.

Doudna J.A.

Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Siksnys et al., 2012 Siksnys, V., Gasiunas, G., and Karvelis, T. (2012). RNA-directed DNA cleavage by the Cas9-crRNA complex from CRISPR3/Cas immune system of Streptococcus thermophilus. U.S. Provisional Patent Application 61/613,373, filed March 20, 2012; later published as US2015/0045546 (pending). Later studies would reveal that tracrRNA also has another key role. Subsequent biochemical studies showed that tracrRNA was not only involved in processing crRNA but was also essential for the Cas9 nuclease complex to cleave DNA ().

Reconstituting CRISPR in a Distant Organism Virginijus Siksnys grew up in Soviet-era Lithuania and graduated from Vilnius University before leaving home in the early 1980s to get a Ph.D. at Moscow State University, where he studied enzyme kinetics. When he returned home to Vilnius, he joined the Institute of Applied Enzymology to study the then-hot field of restriction enzymes. After two decades, though, he was bored with characterizing restriction enzymes. Horvath, Barrangou, and Moineau’s 2007 paper re-ignited his fascination with bacterial barriers to foreign DNA. As a chemist, he felt that he would only understand CRISPR if he could reconstitute it in vitro. Sapranauskas et al., 2011 Sapranauskas R.

Gasiunas G.

Fremaux C.

Barrangou R.

Horvath P.

Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Bolotin et al., 2005 Bolotin A.

Quinquis B.

Sorokin A.

Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Makarova et al., 2006 Makarova K.S.

Grishin N.V.

Shabalina S.A.

Wolf Y.I.

Koonin E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. His first step was to test whether he had all of the necessary components. He and his collaborators set out to see whether the CRISPR system from S. thermophilus could be reconstituted in fully functional form in a very distant microbe, E. coli. To their delight, they found that transferring the entire CRISPR locus was sufficient to cause targeted interference against both plasmid and bacteriophage DNA (). Using their heterologous system, they also proved that Cas9 is the only protein required for interference and that its RuvC- and HNH-nuclease domains () are each essential for interference. The field had reached a critical milestone: the necessary and sufficient components of the CRISPR-Cas9 interference system—the Cas9 nuclease, crRNA, and tracrRNA—were now known. The system had been completely dissected based on elegant bioinformatics, genetics, and molecular biology. It was now time to turn to precise biochemical experiments to try to confirm and extend the results in a test tube.

Studying CRISPR In Vitro Gasiunas et al., 2012 Gasiunas G.

Barrangou R.

Horvath P.

Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Siksnys et al., 2012 Siksnys, V., Gasiunas, G., and Karvelis, T. (2012). RNA-directed DNA cleavage by the Cas9-crRNA complex from CRISPR3/Cas immune system of Streptococcus thermophilus. U.S. Provisional Patent Application 61/613,373, filed March 20, 2012; later published as US2015/0045546 (pending). Using their heterologous expression system in E. coli, Siksnys and his colleagues purified the S. thermophilus Cas9-crRNA complex by using a streptavidin tag on Cas9 and studied its activity in a test tube (). They showed that the complex could cleave a DNA target in vitro, creating a double-stranded break precisely 3 nucleotides from the PAM sequence—matching the in vivo observations of Moineau and colleagues. Most dramatically, they demonstrated that they could reprogram Cas9 with custom-designed spacers in the CRISPR array to cut a target site of their choosing in vitro. By mutating the catalytic residues of the HNH- and RuvC-nuclease domains, they also proved that the former cleaves the strand complementary to the crRNA while the latter cleaves the opposite strand. And, they showed that the crRNA could be trimmed down to just 20 nucleotides and still achieve efficient cleavage. Finally, Siksnys showed that the system could also be reconstituted in a second way—by combining purified His-tagged Cas9, in-vitro-transcribed tracrRNA and crRNA, and RNase III—and that both RNAs were essential for Cas9 to cut DNA. (They would ultimately drop the second reconstitution from their revised paper, but they reported all of the work in their published U.S. patent application filed in March 2012 []). Around the same time, Charpentier had begun biochemical characterization of CRISPR with a colleague in Vienna. When she lectured about tracrRNA at an American Society for Microbiology meeting in Puerto Rico in March 2011, she met Jennifer Doudna—a world-renowned structural biologist and RNA expert at the University of California, Berkeley. After growing up in Hawaii, Doudna had received her Ph.D. at Harvard, working with Jack Szostak to re-engineer an RNA self-splicing intron into a ribozyme capable of copying an RNA template, and had then done postdoctoral work with Tom Cech at the University of Colorado, where she had solved crystal structures of ribozymes. In her own lab (first at Yale in 1994 and then at Berkeley starting in 2002), she characterized RNA-protein complexes underlying diverse phenomena, such as internal ribosome entry sites and processing of microRNAs. She had been using crystallography and cryo-electron microscopy to solve structures of components of the Cascade complex of type I CRISPR systems, the more complex systems used in microbes such as E. coli. Jinek et al., 2012 Jinek M.

Chylinski K.

Fonfara I.

Hauer M.

Doudna J.A.

Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. The two scientists decided to join forces. They used recombinant Cas9 (from S. pyogenes expressed in E. coli) and crRNA and tracrRNA that had been transcribed in vitro (). Like Siksnys, they showed that Cas9 could cut purified DNA in vitro, that it could be programmed with custom-designed crRNAs, that the two nuclease domains cut opposite strands, and that both crRNA and tracrRNA were required for Cas9 to function. In addition, they showed that the two RNAs could function in vitro when fused into a single-guide RNA (sgRNA). The concept of sgRNAs would become widely used in genome editing, after modifications by others to make it work efficiently in vivo. Siksnys submitted his paper to Cell on April 6, 2012. Six days later, the journal rejected the paper without external review. (In hindsight, Cell’s editor agrees the paper turned out to be very important.) Siksnys condensed the manuscript and sent it on May 21 to the Proceedings of the National Academy of Sciences, which published it online on September 4. Charpentier and Doudna’s paper fared better. Submitted to Science 2 months after Siksnys’s on June 8, it sailed through review and appeared online on June 28. Both groups clearly recognized the potential for biotechnology, with Siksnys declaring that “these findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases,” and Charpentier and Doudna noting “the potential to exploit the system for RNA-programmable genome editing.” (A few years later, Doudna would call the world’s attention to the important societal issues raised by the prospect of editing the human germline.)