Release date: 2016-04-05

Three years ago, scientists announced that CRISPR technology enables accurate and efficient genome editing of eukaryotic living cells. Since then, this technology has shaken the scientific community, and thousands of laboratories are applying it to everything from biomedicine to agriculture. However, starting from the discovery of a peculiar bacterial repetitive sequence phenomenon, to confirm that this phenomenon is an adaptive immune system, and then to understand its biological functional characteristics, until the development of a genetic engineering technology, which was previously twenty The relevant research process is not known. This article is aimed at filling the gap in this scientific history. It tells the evolutionary history of ideas and the legendary stories of pioneering characters, and derives enlightenment from the excellent scientific research environment that supports scientific discovery.

Foreword

It's hard to remember which science revolution once changed the biology world so quickly like CRISPR. Just three years ago, scientists announced that the CRISPR system, the adaptive immune system that bacteria can defend against by invading the DNA sequence of the invading virus, can be transformed into a simple and effective technique for mammals and other organisms. Genome editing in the living cells of the body. This is why thousands of laboratories around the world are used in a wide range of fields, such as the creation of complex animal models of human genetic diseases and cancer; genome-wide screening in human cells to pinpoint specific genes that act on physiological processes; Or shut down the role of a particular gene; alter the genes of the plant. The prospect that CRISPR has the potential to change the human reproductive system has sparked a global debate.

Although I haven't seen molecular biologists who haven't heard of CRISPR, if you ask them how this scientific revolution happened, they tend to be confused. Immunologist Peter? Sir Peter Medawar has a cloud: “The history of science makes most scientists feel bored” (Medawar, 1968). Indeed, scientists always focus on the future without hesitation. Once a fact is scientifically established, the path leading to the discovery of this fact is classified as an irrelevant anecdote.

However, the story of scientists behind scientific breakthroughs can give us more insight into this magical research environment that is good at inspiring biomedical advancement: inspiration and planning, pure curiosity and practical use, “Hypothesis-free ) and "hypothesis-driven" research methods, individuals and teams, novel perspectives and deep expertise in which to play their respective functions. These understandings are especially important for governments and foundations, because in the United States alone, these organizations have invested more than $40 billion in biomedical research. These understandings are equally important to the general public who often think of scientists as lonely geniuses who live in isolation from the laboratory. In addition, for science apprentices who are undergoing scientific training, it is very beneficial to have a realistic picture of the scientific career and to use it as a guide and incentive.

In the past few months, I have been trying to understand the 20 years of past events behind CRISPR, including the history of scientific ideas and the personal experiences of scientists. The perspective of this paper is based on published papers, personal interviews and other materials (including journal rejection letters). Finally, I tried to draw some general experience from it. (As a background, Figure 1 provides a brief overview of the Type II CRISPR system, which is transformed into genome editing.) The key element of this paper is to describe a group of smug scientists along with their collaborators and others. The contributors who can be detailed have discovered the CRISPR system, unveiled its molecular mechanism, and transformed it into a powerful tool for biological and biomedical research. They are listed together in the CRISPR hero spectrum.

CRISPR is found

The story begins in the Mediterranean port of Santa Pola on the White Coast of Spain, where the beautiful coast and vast salt marshes have attracted holidaymakers, flamingos and salt producers for centuries (the geography of this story) as shown in picture 2). Francisco Mojica grew up nearby and frequented the beach. Naturally, when he started his Ph.D. study at the University of Alicante on the upper coast in 1989, he joined a study on Mediterranean halophila. The laboratory of Haloferax mediterranei, an archaea with extreme salt tolerance isolated from the marsh of St. Paula. His mentor found that the salt content of the medium appeared to affect the restriction enzymes that cleave the microbe's genes, and Mojica began to identify this heterologous fragment. In the first DNA fragment he examined, Mojica discovered a strange structure, a nearly perfect, roughly palindrome, 30-base, multi-copy repeat separated by 36 bases. Sequence, which is different from the family of repeats of any known microorganism (Mojica et al., 1993).

The 28-year-old graduate student was deeply attracted to this and contributed his next decade of academic career to the mystery of cracking. He soon found similar repeats in the similar H. volcanii and the more distant halophilic archaea. In the process of combing the scientific literature, he discovered the association of this phenomenon with eubacteria: a paper by a Japanese research group (Ishino et al., 1987) mentioned a repetitive sequence in Escherichia coli. A similar structure, although there is no sequence similarity to the repetition of Halofarax. The authors of the paper did not delve into this phenomenon, but Mojica realized that the existence of such a similar structure on a microbe with such a relationship must imply an important function in a prokaryotic cell that has not yet been discovered. Before going to Oxford for a short postdoctoral study, he wrote a paper covering this new repetitive sequence category (Mojica et al., 1995).

Mojica then returned to the University of Alicante to serve as a faculty member. Because the school lacked the launch of research funding and laboratory space, he had to turn to bioinformatics to study this strange repetition and named it short regularly spaced repeats (SRSPs). This name was later changed to "Clustered Regularly Interspaced Palindromic Repeats, CRISPR" under his own suggestion (Jansen et al., 2002; Mojica and Garrett, 2012).

By 2000, Mojica had discovered CRISPR loci on 20 different microorganisms, including Mycobacterium tuberculosis, Clostridium difficile, and Yersinia pestis (Mojica et al. 2000). . In two years, researchers have doubled the list of related microbes and listed key features of the locus, including the presence of close relatives of specific "CRISPR-associated genes" (cas genes), which are generally considered Their function is related (Jansen et al., 2002). (Listing 1 lists the latest classifications of the CRISPR system.)

But what is the function of the CRISPR system? Various assumptions emerge in an endless stream: for example, it is related to gene regulation, replication partitioning, DNA repair, and other functions. However, most of these assumptions are not supported by evidence, and they are all falsified one by one. As with the discovery of CRISPR, important insights come from bioinformatics.

CRISPR is an adaptive immune system

During the holiday in August 2003, Mojica avoided the heat of Santa Pola and hid in the air-conditioned office in Alicante. Nowadays, as the leader of the emerging field of CRISPR, he has turned his attention from repeating sequences to separating their interval sequences. Using a word processor, Mojica worked tirelessly to extract each interval and insert it into the BLAST software to search for similarities to any other known DNA sequence. Although he tried this method failed, the DNA sequence database is expanding, and this time he successfully dig into the gold mine. On a CRISPR gene locus that he recently sequenced from an E. coli strain, one of the spacers matches the sequence of a P1 phage, and this phage can infect a variety of E. coli strains. However, strains carrying this interval are known to be resistant to P1 infection. At the end of the week, he had retrieved 4,500 intervals. Of these, 88 intervals are similar to the known sequences, and two-thirds match the virus-associated virus or ligated plasmid carrying the spacer. Mojica recognizes that the CRISPR locus stores the information needed for an adaptive immune system to protect microorganisms against infection.

Mojica then went out to drink with cognac and celebrated with her colleagues, and began writing relevant papers early the next morning. So I started the pain of suffering for 18 months. Recognizing the importance of this discovery, Mojica voted for Nature. The publication of the paper rejected by the journal in November 2003 without an external review. It is difficult to understand that the editor's key arguments claiming the paper belong to the known category. In January 2004, the decision of the Proceedings of the National Academy of Sciences was that the paper lacked “sufficient novelty and importance” and was therefore not qualified for trial. Molecular Microbiology and Nucleic Acid Research have also refused to publish. At this time, Mojica, desperate and worried about being one of the first to be published, voted for the Journal of Molecular Evolution. After more than 12 months of review and revision, this paper, which announced the possible functions of CRISPR, was finally published on February 1, 2005 (Mojica et al., 2005).

At the same time, CRISPR is the focus of researchers in another unexpected location, a research department of the French Ministry of Defense 30 km south of Paris. Giles Vergnaud is a human geneticist trained at the Pasteur Institute, and his Ph.D. and postdoctoral research is funded by the French Armaments Directorate. After completing his postdoctoral research in 1987, he joined the French Ministry of Defense and established its first molecular biology laboratory. In the next decade, Vergnaud continued his research in human genetics. However, after intelligence agencies raised concerns about the development of biological weapons by Iraq’s Saddam Saddam regime in the late 1990s, the Department of Defense in 1997 asked Vergaud and his team to shift their research focus to forensic microbiology. This development is based on the microgenetic differences between microbial species to track the source of the pathogen. In a joint laboratory with the nearby Institute of Genetics and Microbiology at the University of Paris, he began using tandem repeat polymorphism, the main tool of this forensic human DNA fingerprinting, to map to anthrax (anthrax) And the bacterial species of the plague.

The French Ministry of Defence has a special sample of 61 plague bacilli from the plague outbreak in Vietnam from 1964 to 1965. Vergnaud found that these closely related isolates were identical at the tandem repeat locus, with the exception of one position, the CRISPR locus discovered by his colleague Christine Pourcel. Their strains are distinguished by occasional new intervals that are not at the anterior end of the CRISPR locus (Pourcel et al., 2005). It is worth noting that many of these new compartments match the prophage present on the Yersinia pestis gene. The authors concluded that the CRISPR locus is performing a defense mechanism, in poetic language, that is, "CRISPR may reproduce the memory of past genetic attacks." Vergnaud tried to publish the efforts they found and Mojica encountered the same Obstruction. The paper was rejected by the Proceedings of the National Academy of Sciences, the Journal of Bacteriology, Nucleic Acid Research, and Genomics Research until it was published on March 1, 2005 in Microbiology.

Finally, the third researcher Alexander Bolotin, a Russian microbiologist at the French National Institute of Agricultural Research, also published a paper on the origin of CRISPR originating from chromosomes in the September 2005 issue of Microbiology (Bolotin et al. 2005). Since his paper was previously rejected by another journal, it was actually submitted one month after Mojica's February 2005 paper was published. It is worth noting that Bolotin first proposed the idea of ​​how CRISPR provides immune function. He speculates that transcripts from the CRISPR locus work by antisense RNA inhibition of phage gene expression. Although this assumption sounds reasonable, it will soon be falsified.

The experimental evidence that CRISPR provides adaptive immune function and uses nucleases is like Mojica, and Philippe Horvath may not find a topic that is more local or more boring. As a Ph.D. student at the University of Strasbourg, he studied lactic acid bacteria used in the production of sauerkraut. This pickled cabbage is Alsatian. The main ingredient of the dish pickled cabbage pork potatoes (choucroutegarnie). Out of his interest in food science, Horvath skipped postdoctoral research and joined Rhodia Food in 2000, a bacterial starter production in Dange-Saint-Romain in western France. Business, where the company established the first molecular biology laboratory of the company. The company was later acquired by the Danish company Danisco, and Danisco was acquired by DuPont in 2011.

Rhodia Foods is interested in Horvath's microbiology expertise because other lactic acid bacteria such as Streptococcus thermophiles have been used in the production of dairy products such as yogurt and cheese. Horvath's goals include the development of DNA-based techniques to accurately identify strains and overcome frequent phage infections, a dysentery that plagues industrial starter production for dairy fermentation. Therefore, understanding the mechanism by which specific S. thermophilus strains resist self-protection against phage attack has both scientific and commercial value.

After learning about CRISPR at a Dutch conference on lactic acid bacteria in the second half of 2002, Horvath began using this method to identify the genotype of his strain. In the second half of 2004, he noticed a correlation between the interval and resistance to phage, and the same findings were published by Mojica and Vergnaud a few months later. In 2005, Horvath and his colleagues, including Rodolphe Barrangou, a newcomer to Dennis Co., USA, and Sylvain Moineau, a famous phage biologist at Universe Laval in Quebec City, set out to test CRISPR. It is an assumption of an adaptive immune system. Coincidentally, Moineau was also a scientist in the industrial field. He obtained his Ph.D. in food science from Laval, studied lactic acid bacteria, and worked at Unilever Corporation before returning to Laval as a faculty member. He has worked with Rhodia Foods since 2000.

Using a phage-sensitive strain of Streptococcus thermophilus and two phages, the researchers used genetic screening to isolate phage-resistant strains. Rather than containing traditional resistance mutations (such as mutations on cell surface receptors required for phage invasion), this resistant strain acquires phage-derived sequences at their CRISPR loci (Barrangou et al. 2007). In addition, multiple spaced insertions are associated with enhanced resistance. It is in this process that they have acquired immune function.

They also studied the effects of two of the cas genes: cas7 and cas9. Bacteria require cas7 to gain resistance, but these gaps that carry the phage source do not require this gene to maintain resistance, suggesting that cas7 assists in creating new intervals and duplications, but it does not involve the immune function itself. In contrast, cas9 is essential for resistance to phage because its sequence contains two nuclease modules (HNH and RuvC) and its products are then thought to cleave nucleic acids (Bolotin et al., 2005; Makarova). Et al., 2006); and, cas9 protein is an active component of the bacterial immune system. (Hint: In the early CRISPR literature, the now famous cas9 gene is called cas5 or csn1.) Finally, they found that rare phage isolates that overcome CRISPR-based immune functions carry a single base change in their genes. This changes the sequence that would otherwise match the interval. Thus, immune function is achieved by relying on precise DNA sequence matching between the interval and the target.

Design CRISPR

John van der Oost received his Ph.D. from the Free University of Amsterdam in 1989. He was originally interested in solving the world's clean energy needs and studying how to use cyanobacteria to produce biofuels. Before returning to Amsterdam, he studied the metabolic pathways of bacteria in Helsinki and Heidelberg. In 1995, Wageningen University issued a tenure appointment to him, but only if he needed to grow a team that specializes in microbes that survive extreme conditions. Van der Oost has heard about Streptococcus thermophilus that can multiply in the hot springs of Yellowstone National Park in Germany, so he is eager to explore the evolutionary differences in the metabolic pathways of these exotic microorganisms. He began working with Eugene Koonin, an expert in microbial evolution and computer biology at the National Center for Biotechnology Information (NCBI) at the National Institutes of Health. Koonin has already begun to classify and analyze the CRISPR system. In an interview in 2005, he led van der Oost to the little-known CRISPR field (Makarova et al., 2006).

Van der Oost received major funding from the National Science Foundation of the Netherlands. He decided to use part of the funds for research in addition to the research topic. (In a report five years later, he emphasized the usefulness of the above-mentioned institution's policy of giving researchers the freedom to change the direction of the research program.) He and his colleagues embed an E. coli CRISPR system into another lack of this self. E. coli strain of endogenous system. This allows them to biochemically identify a complex of five cas proteins called Cascade (Brouns et al., 2008). (E. coli has a more complex class 1, type I CRISPR system, in which the function of cas9 is achieved by the Cascade complex in conjunction with the nuclease cas3. See Listing 1.) By culling each component one by one, they prove Cascade is required for accessing a long precursor RNA transcribed via a CRISPR locus into a 61 nucleotide long CRISPR RNA (crRNAs). After cloning and gene sequencing of a set of crRNAs purified with the Cascade complex, they were all started with an 8-base repeat, followed by a complete interval and a new repeat. This finding supports the previous hypothesis that the palindromic structural properties of the repeat sequence result in the formation of secondary structures in the crRNA (Sorek et al., 2008).

To demonstrate that crRNA sequences are responsible for generating CRISPR-based resistance, they set out to create a first-person CRISPR arrangement that sets CRISPR to target four basic genes for lambda phage. As they expected, lines carrying the new CRISPR sequence were resistant to phage. This is the first program-based, CRISPR-based immunization ever made, like a flu vaccine for bacteria.

These experimental results suggest that the goal of CRISPR is not RNA (Bolotin conceived) but DNA. The researchers designed two versions of the CRISPR array, one in the antisense strand orientation (complementing the coding strands of the mRNA and DNA sites) and the other in the sense strand orientation (complementary to another DNA strand). Although the interval differs in effectiveness, the fact that the experiment works in the version of the sense chain direction strongly suggests that the target is not mRNA. However, it is not direct evidence. Under the editorial of Science, who insisted on a firm judgment on the paper, van der Oost put the idea of ​​using CRISPR as a target in a "guess" way.

The target of CRISPR is DNA

Luciano Marraffini is completing a Ph.D. study at the University of Chicago with a research focus on Staphylococcus, where she learned about CRISPR from the world's leading phage genetics authority, Malcolm Casadaban. In 2005, Casadaban immediately saw that CRISPR is likely to be an important part of the adaptive immune system, and that everyone who is interested in it talks about CRISPR. Like many scientists in the field of phage research, Marraffini believes that CRISPR is not affected by RNA interference, because this mechanism is powerless to overcome the explosive growth that occurs during phage infection. He concluded that CRISPR must cut the DNA, and this function is like the role of restriction enzymes.

Marraffini was eager to join the few research teams in the world who are studying CRISPR for postdoctoral research, but because his wife has an interpreter at the Criminal Court in Cook County, Illinois. Good job, he feels he has to stay in Chicago. He persuaded Erik Sontheimer, a biochemist at Northwestern University, to join his laboratory to study CRISPR, which has been working on RNA splicing and RNA interference.

Before moving to Northwestern University, Marraffini started his Ph.D. research and started working on CRISPR to see if the Staphylococcus CRISPR system can prevent plasmid junctions. He noted that one Staphylococcus epidermidis possesses an interval that matches a region of the nickase (nes) gene stored on a plasmid from antibiotic-resistant Staphylococcus aureus. He demonstrated that these plasmids were unable to transform S. epidermidis, and that disruption of either the nes sequence in the plasmid or its mating interval at the CRISPR locus would cancel the interference function (Marraffini and Sonthheimer, 2008). Obviously, CRISPR blocks the plasmid as if it were a virus.

Marraffini and Sonthheimer once considered recombining the CRISPR system in vitro to prove that it can cleave DNA. But the S. epidermidis system is too complex, it has 9 cas genes, and its genetic characteristics are completely unmastered. So they turned their attention to molecular biology. They cleverly changed the nes gene in the plasmid directed against the target of the CRISPR system by embedding a self-splicing intron in the middle of its sequence. If CRISPR is targeted for mRNA, then this change will not affect the interference function because the intron sequence will be clipped. And if CRISPR is targeted at DNA, then this embedding will cancel the interference function because the intervals will no longer match. The result is clear: the goal of CRISPR is DNA.

Marraffini and Sonthheimer recognize that CRISPR is essentially an editable set of restriction enzymes. Their paper is the first to clearly present a prediction: CRISPR can be used to perform genome editing in a heterogeneous system. “From a practical point of view,” they announced, “the ability to orientate DNA containing any known 24- to 48-nucleotide target has considerable functional utility, especially if the system It can also function outside of the original bacterial or archaeal environment.” They even filed a patent application involving the use of CRISPR to cleave and correct genetic loci in eukaryotic cells, but eventually abandoned the application due to lack of experimental evidence (Sontheimer And Marraffini, 2008).

Cas9 is directed by crRNAs and produces double-strand breaks in DNA

After far-reaching research in 2007 confirmed that CRISPR is an adaptive immune system (Barrangou et al., 2007), Sylvain Moineau continues to work with Denisco to understand the mechanism by which CRISPR cleaves DNA.

The problem is that CRISPR is always so efficient under normal circumstances, making it impossible for Moineau and colleagues to easily see how invading DNA is destroyed. However, they were favored by the goddess of fortune during the study of plasmid interference by Streptococcus thermophilus. The researchers found a small number of strains whose CRISPRs only provide partial defense against plasmids that rely on electroporation. In the cells of one of the inefficient lines, it can be seen that the linear plasmid is still present. To some extent, the process of plasmid interference can be slowed down so that the products of CRISPR activity can be observed (Garneau et al., 2010).

This line allowed them to study the cutting process. Consistent with their previous results (Barrangou et al., 2007), this time the results show that plasmid cleavage is dependent on cas9 nuclease. When sequencing linearized plasmids, they found a single, precisely blunt-cut trinucleotide upstream of the PAM (proto-spacer adjacent motif), a key sequence feature that was previously described in the paper. The role has been described (Deveau et al, 2008; Hovath et al, 2008). After extending the scope of the analysis, the results showed that the DNA of the virus was also precisely cut at the same position associated with the PAM sequence. In addition, the number of different intervals matching the same target is consistent with the number of cuts.

The experimental results clearly indicate that the nuclease activity of Cas9 is to cleave DNA at precise points, which are encoded by the specific sequence of the crRNA.

TracrRNA discovery

Even under the intensive study of the CRISPR-Cas9 system, the puzzle that makes up this puzzle is still missing, a small RNA that was later called the trans-acting CRISPR RNA (tracrRNA). In fact, its discoverers Emmanuelle Charpentier and J?rg Vogel did not specifically study the CRISPR system; they only attempted to identify microbial RNA.

Charpentier received his Ph.D. from the Pasteur Institute in 1995, followed by a six-year postdoctoral fellowship in New York, followed by the University of Vienna in 2002 and Umeå University in Sweden in 2008 (Ume? University) establishes its own laboratory. After she discovered an unusual RNA that controls her infectivity in Streptococcus pyogenes (Mangold et al., 2004), she became interested in identifying more regulatory RNA in microbes. She used the bioinformatics program to search for the gene span of S. pyogenes, envisioning that they might encode non-coding RNA. She found several candidate intervals, including one near the CRISPR locus, but lacking direct information about these RNAs is too difficult to study.

When Charpentier met Vogel at the 2007 RNA Society in Madison, Wisconsin, the solution came into being. Vogel, a microbiologist trained in Germany, began specializing in RNA searching for pathogens in post-doctoral studies in Uppsala and Jerusalem. This work continued until 2004 when he was infected with a horse in Berlin. When the Institute established its own research team. (After five years, he moved to Würzburg to lead an infectious disease research center.) With the advent of "next-generation sequencing" technology, Vogel realized that massively parallel sequencing would enable the mapping of any microbial transcriptome Recording is possible. He had used this method at the time for the bacteria that caused the stomach ulcer, Helicobacter pylori (Sharma et al., 2010), and was being used for other bacteria. Charpentier and Vogel decided to target S. pyogenes.

This method produced a surprising result: the third most abundant RNA transcript after ribosomal RNA and transporter RNA belongs to a new class of small RNAs that are close to the CRISPR locus (that is, that The sequence of the Chapentier note is transcribed, and it has 25 bases that are nearly perfectly complementary to the CRISPR repeat. This complementarity indicates that the tracrRNA and the precursor of the crRNA hybridize together and are cleaved by RNase III to become a mature product. Genetic deletion experiments have also confirmed the notion that tracerRNA is essential for the processing of crRNA and is therefore essential for CRISPR action (Deltcheva et al., 2011).

Subsequent research revealed that tracerRNA has another key role. Subsequent biochemical studies have shown that tracer RNA is not only involved in the processing of crRNA, it is also required for the Cas9 nuclease complex to cleave DNA (Jinek et al., 2012; Siksnys et al., 2012).

Reconstruction of CRISPR within distant species

Virginijus Siksnys grew up in Lithuania in the Soviet era. After graduating from Vilnius University, he went to Moscow State University in the early 1980s to study his kinetics. After that, he returned to his hometown of Vilnius to join the Institute of Applied Enzymes, where he worked on the popular restriction enzymes field. Twenty years later, he was already tired of studying restriction enzymes. Horath, Barrangou and Moineau's 2007 paper rekindled his interest in the defense of bacteria against external DNA. As a chemist, he realized that to understand CRISPR, he had to rebuild it in vitro.

His first step is to test if he has acquired all the necessary components. He and his collaborators began to observe whether the CRISPR gene locus from S. thermophiles could be reconstituted in a distant microbial E. coli into a fully functional form. To their delight, they found that translocation into the entire CRISPR locus was sufficient to achieve targeted interference with plasmid and phage DNA (Sapranauskas et al., 2011). They also demonstrated that Cas9 is the only essential protein for interference activity using a heterologous system, and that its Ruvc and HNH nuclease domains are essential.

As the necessary and sufficient components of the CRISPR-Cas9 interfering system, Cas9 nuclease, crRNA and tracrRNA, have been discovered, research in this area has reached a critical milestone. This system has been completely mastered by cutting-edge bioinformatics, genetics and molecular biology. It is time to turn the direction to precise biochemical experiments to confirm and extend these conclusions in test tubes.

Study CRISPR in test tubes

Using their heterologous expression system in E. coli, Siksnys and colleagues purified the Cas9-crRNA complex of S. thermophilus by labeling Cas9 with a streptavidin label and observed its activity in vitro ( Gasiunas et al., 2012). The results showed that the complex was able to cleave the DNA target in vitro, creating a double-strand break that is exactly three nucleotides away from the PAM sequence—just as it is observed in the bacteria in Moineau and colleagues. Most importantly, experiments have shown that they can adapt Cas9 to a specially designed spacer sequence in the CRISPR array to achieve a cut in the test tube for the selected target position. By transforming the catalytic residues of the HNH and RuvC nuclease domains, they also demonstrated that the former cleaves the gene strand complementary to the crRNA, while the latter cleaves the opposite strand. They also demonstrated that crRNA can be trimmed to only 20 nucleotides remaining while still maintaining effective cutting power. Finally, Siksnys proved that this system can also be reconstructed using the second method—that is, the purified His-tagged Cas9, the tracrRNA transcribed in vitro and the crRNA, and the RNase III—the two RNAs are cleaved for Cas9. DNA is essential. (Ultimately they removed the second reconstruction method in the revised paper, but reported all the research content in their published US patent application filed in March 2012 [Siksnys et al., 2012]).

At the same time, Charpentier has begun a biochemical identification of CRISPR with a colleague from Vienna. When she reported her research on tracrRNA at the American Society of Microbiology conference in Puerto Rico in March 2011, she met Jennifer Doudna, a world-renowned structural biologist and RNA specialist at the University of California. Doudna, who grew up in Hawaii, received his Ph.D. from Harvard University and worked with Jack Szostak to reshape an RNA self-splicing intron to a ribozyme with the ability to replicate RNA templates. She later solved the crystal structure of ribozymes after doing postdoctoral research with Tom Cech of the University of Colorado. In her own laboratory (founded in Yale in 1994 and then in Berkeley in 2002), she identified RNA protein complexes under various phenomena, such as the location of internal ribosomes and the processing of microRNAs. . She has been using crystallography and cryo-electron microscopy to solve structural problems in the components of the Cascade complex of the Type I CRISPR system, a more complex system such as E. coli.

The two scientists decided to join forces. They used recombinant Cas9 (a gene from S. pyogenes expressed in E. coli) and in vitro transcribed crRNA and tracrRNA (Jinek et al., 2012). Like Siksnys, they also demonstrated that Cas9 can cleave purified DNA in vitro. It can be designed with specially designed crRNA. The two nuclease domains cleave the opposite two DNA strands, respectively, and the crRNA and tracrRNA play against Cas9. The role is required.此外，她们证明两种RNA在被融合为单一的导向RNA（sgRNA）时也可以在体外发挥作用。在经过其他科学家的修改而变得可以更高效地在体外作用之后，sgRNA的概念在基因组编辑领域被广泛使用。

Siksnys于2012年4月6日向《细胞》提交了论文。6天之后，在没到外部评审的情况下就被期刊拒绝了。（事后，《细胞》的编辑承认这篇论文其实是非常重要的。）Silsnys于是做了此论文的精炼版本，于5月21日将其投稿给了《美国国家科学学院院刊》，得以于9月4日在线发表。Charpentier和Doudna的合作论文的运气则要好得多。在Siksnys的论文提交之后的2个月，她们的论文于6月8日提交给了《科学》，顺利通过评审并于6月28日在线发表。

两组研究团队都清楚地认识到CRISPR对于生物技术的潜在价值。Siksnys宣称：“这些发现为构造普遍可设计的RNA引导的DNA核酸内切酶铺平了道路。”而Charpentier和Doudna则提到：“利用这一系统来进行可设计的基因组编辑的潜力。”（几年之后，Doudna让世界的注意力里投向编辑人类生殖系统这一前景所引起的重要社会性问题。）在哺乳动物细胞内进行基因组编辑。

在1980年代后期科学家们设计出一种可以在活细胞内改变哺乳动物基因的方法，这彻底改变了生物医学研究，包括使得在老鼠的胚胎干细胞内的特定位置嵌入DNA并且培育出携带这一遗传改变的后代成为可能（综述见Capeccchi，2005）。虽然这个方法是革命性的，但是过程却是低效的，因为它需要通过筛选识别出那些百万分之一的细胞，就是在其内通过同源重组与由实验者提供的修改过的版本之间交换了一个基因。1990年代中期，哺乳动物学家在酵母遗传学的观察基础上发现在某个基因位点上引进一个双链断裂可以极大地增强同源重组和由非同源末端接合导致的小缺失的发生频率（综述见Haber，2000和Jasin与Rothstein，2013）。他们意识到，高效基因组编辑的秘诀在于找到一种可以在任何想要的位置制造出双链断裂的可靠手段。最初的普遍策略是使用锌手指核酸酶（ZFNs）——一种由一个锌手指DNA结合域和一个取自限制性内切酶的DNA切割结构域所组成的融合蛋白，它可以结合并切割基因组位点（Bibikova 等，2001）。不久科学家们就在果蝇和小鼠身上证明了ZFNs依靠同源重组对于具体位置上的基因组编辑的用途（Bibikova 等，2003；Porteus和Baltimore，2003）。到2005年，桑加莫生物科学公司（Sangamo Biosciensces）的研究小组报告了针对在人类细胞系上造成重度联合免疫综合症的基因的突变进行了成功的修正（Urnov 等，2005）。然而，塑造能够可靠辨认具体位置的ZFNs的过程是缓慢而费力的。更好的方法则出现在2009年下半年，两个研究小组描述了一组来自于植物病原体黄单胞菌（Xanthomonas）（Boch 等，2009；Moscou和Bogdanove，2009），叫做TALEs的特殊转录激活蛋白，TALE使用一个特定的模块密码来瞄准具体的DNA序列。但是，这一方法还是需要可观的工作量，因为针对每个目标都要求设计一个新的蛋白。

自从Marraffini和Sontheimer的2008年论文证明了CRISPR是一种可设计的限制性内切酶以来，研究人员已经意识到如果它可以在哺乳动物细胞内发挥作用的话，CRISPR也许就能为具体基因位点的切割和编辑提供强大的工具。但是，这个“如果”是非常关键的。相对于微生物，哺乳动物细胞拥有不同的内部环境，它们的基因要大1000倍，位置在细胞核内，并且嵌在一个精致复杂的染色质结构内。转入诸如自我剪接II类内含子的其它简单微生物系统已经失败了，而使用核酸来瞄准基因位点的尝试也问题重重。CRISPR究竟能否被设计构造成为一种用于编辑人类基因组的强力工具？直到2012年9月，专家们是持怀疑态度的（Barrangou，2012； Carroll, 2012）。

张锋11岁时从中国石家庄搬到爱荷华州的得梅因（Des Moines, Iowa）。他在一次周六兴趣课程中被分子生物学深深吸引，在16岁时就在一家当地的基因疗法实验室每周工作20小时。在哈佛念本科的时候，他因为室友患了重度抑郁症的事而开始对大脑发生兴趣，后来他到斯坦福跟随神经生物学家兼精神病学家Karl Deisseroth攻读博士学位，期间他们（联同Edward Boyden）发展出了光遗传学（optogentics），这是一项革命性的技术，依靠此技术可以通过光束来触发携带微生物光敏感通道蛋白的神经元。在波士顿身为独立研究员期间（先后在哈佛和麻省理工学院的脑及认知科学系还有博德研究所［Broad Institute］作初级研究员），张致力于进一步扩展研究神经生物学的分子工具。在开发出一种使用光来激活基因表达（通过把一个DNA结合结构域和一个转录激活结构域同两个在光条件下相互结合的植物蛋白偶联）的方法之后，他开始探索一种用来设计转录因子的普遍方法。在TALEs被解码之后，张与他的合作者Paola Arlotta和George Church（此外还有一个来自桑加莫生物科学公司的研究小组）都成功地将TALEs改造用于哺乳动物，使得精准激活、抑制和编辑基因成为可能（张等，2011；Miller等，2011）。然而，张锋并没有停止寻找更好的方法。

2011年2月张听了哈佛的微生物学家Michael Gilmore关于CRISPR的报告就立刻被它吸引住了。他次日飞赴迈阿密参加一个学术会议，却闷在酒店房间里一头扎进CRISPR的文献资料。回来之后，他就着手创造嗜热链球菌的Cas9版本用于人类细胞（用优化密码子和一个核定位信号）。到2011年4月，他已经发现，通过表达Cas9基因和一个设计过的靶向携带荧光素酶的质粒的CRISPR RNA，他可以在人胚胎肾细胞（HEK）内降低荧光度。但是，效果还是不尽如意。

接下来的一年间张一直在优化这个系统。他探索着可以增加进入细胞核的Cas9比例的方法。当他发现嗜热链球菌Cas9在细胞核内并非平均分布（它在核仁处聚集），他就测试其他的选择，发现化脓性链球菌（S. pyogenes）的Cas9分布地更均匀。他发现哺乳动物细胞虽然缺乏细菌的RNaseIII，但是仍然可以处理crRNA，尽管是用和在细菌里不同的方式。他测试了tracrRNA的各种同工型（isoform）来造鉴定在人类细胞内保持稳定的那一个。

到了2012年的中期，他已经获得了一个由三部分组成的强有效的系统，包含来自化脓性链球菌和嗜热链球菌两者之一的Cas9、tracerRNA和CRISPR阵列。以人和小鼠基因的16个位置为靶向，他证明了，高效而准确地改变基因是可能的——通过非同源重组的末端接合制造缺失和通过与修复模版进行同源重组来实现插入新序列。此外，多重基因可以通过设计CRISPR阵列，用彼此匹配的间隔来同时编辑。当Charpentier和Doudna的论文在那年初夏发表之时，他还用在她们的实验里描述的短sgRNA融合来测试一个两部分组成的系统。在体内环境下这一融合的效果很差，仅仅低效地切割了位点的一小部分，但是，他发现一个复原了3'端发夹结构的全长融合可以解决问题（Cong 等，2013；Zhang，2012）。（张之后很快继续证明了CRISPR比原想的更加功能多样：它可以被用于在数周内创造遗传疾病和体细胞癌的复杂小鼠模型，也可以用于实施全基因组筛选来寻找某一生理过程中的必须基因——它的精确度还可以通过降低脱靶切割来提高。他和曾与van der Oost共事的计算机生物学家Koonin还将发现新的第2类CRISPR系统，包括那个与Cas9切割方式不同而且仅需要crRNA却不需要tracrRNA的核酸酶系统［Zetsche 等，2015］。）张于2012年10月5日提交了报告哺乳动物基因组编辑的论文，发表在2013年1月3日的《科学》上（Cong 等，2013）；这篇论文将成为该领域内被引用次数最多的论文，而他的试剂由一家非营利性组织Addgene分发，在之后的3年间接受到25000次申请。

大约一个月后的10月26日，一个在基因组学和合成生物学领域极有专长并且曾与张有合作的才华横溢而非同寻常的哈佛大学资深教授George Church递交了他关于在人体细胞内进行基因组编辑的论文。自从1970年晚期跟随DNA测序先锋Walter Gibert在哈佛读研究生期间，Church就专注于发展大规模“阅读”和“书写”基因的强大技术——此外他还因具有挑衅性的建议，譬如使用合成生物学来复活长毛的猛犸象（wooly mammoths）和尼安德特人（Neanderthals）而引起社会争论。清楚张的研究，同时受到Charpentier和Doudna的启发，Church着手在哺乳动物细胞内测试crRNA-tracer融合。和张一样，他也发现短融合在体内环境是无效的，而全长融合则效果好。他瞄准7个位置，证明了非同源重组末端接合和同源重组的作用。他的论文和张的论文是相继发表的（Mali 等，2013）。（Church和其他研究者都在不久之后使用CRISPR来提升“基因驱动力”（gene drive）——合成基因在自然群体中可以迅速扩散，从而引发将其应用于控制携带疟疾的蚊子的惊喜和对于破坏生态系统的担忧。他还将寻求使用CRISPR在猪的基因组里灭活逆转录病毒来推动猪的器官移植给人。）到了2012年的夏末，随着体外研究获得瞩目，而成功的体内基因组编辑的消息在发表之前就传播开来，好几个团队开始竞相开展基因组切割的概念验证实验，尽管不是基因组编辑。Doudna在Church的协助下提交了一篇论文，证明在一个基因组位点进行低效率的切割（Pandika 2014；Jinek 等，2013）。曾从事ZFNs和TALEs基因组编辑的韩国首尔国立大学（Seoul National University）的Jin-Soo Kim 教授报道了在两个位点进行切割（Cho 等，2013）。在以上两个例子中，切割都是低效的，因为sgRNA缺乏tracerRNA的关键性3'端发夹结构。作为使用ZFNs和TALEs进行基因组编辑的领军人物，哈佛大学教授Keith Joung 则更进了一步。使用由他的合作者Church提供的全长sgRNA结构，Joung通过在斑马鱼的实验证实CRISPR可以在生殖系上有效地制造缺失（Hwang 等，2013）。这些短论文提交于2012年末，在张和Church的论文2013年1月初发表不久之后被接收，于1月末在线发表。

CRISPR迅速蹿红

2013年初，用谷歌搜索“CRISPR”开始井喷，这一趋势至今未减弱。在一年之内，研究人员已经报告了在多种生物内使用基于CRISPR的基因组编辑，包括酵母、线虫、果蝇、斑马鱼、小鼠和猴子。科学和商业上对其在人类医疗和农业生产的潜在应用的兴趣也开始上升，同时这个技术可能被用来培育设计婴儿的前景也引发了社会关注。

CRISPR的早期开拓者并未停止拓展边界，但是他们不再孤单。全世界的科学家都涌入了这一领域，他们是一批新的英雄队伍，他们进一步地阐明CRISPR的生物学，推进和扩展了基因编组辑技术，并且将它运用于广泛范围的生物学问题。在这篇文章的范围内公正描述他们的贡献是不能的；读者可以参考最近的综述（Barrangou和Marraffini，2014；Hsu 等，2014；van der Oost 等，2014； Sander和Joung，2014；Jiang和Marraffini，2015；Sternberg和Doudna，2015；Wright 等，2016）。

发现20年前的一处西班牙盐沼而曾经不为人知的微生物学系统如今却成了科学期刊特刊、《纽约时报》的头条、生物科技创业公司和国际伦理学峰会的焦点。CRISPR的时代已经到来了。

CRISPR的启迪

CRISPR的故事充满了关于产生科学进步的人文环境的启迪，与学术资助机构、一般公众和踌躇满志的研究人员相关。

其中最重要的一点是医学突破往往来源意想不到的地方。CRISPR的早期英雄们都不是专门研究编辑人类基因的，甚至都是不是研究人类疾病的。他们的动机混合了个人兴趣（弄清楚耐盐细菌内奇怪的重复序列）、军事上的特殊需要（防范生物战争）和工业应用（提高酸奶产量）。

这段历史还说明了基于大数据的“无假设驱动”式发现在生物学研究中愈发重要的位置。CRISPR基因位点，它的生物学功能和tracrRNA的发现都不是来自实验台上的实验，而是来自对大范围、常常是公开的基因数据库的生物信息学的开放式探索。“假设驱动”科学当然依旧是基本的研究方法，但是21世纪将会看到更多的是两种科学研究方法的结合。

如此众多的CRISPR英雄们都在科学生涯的开头（包括Mojica，Marraffini Charpentier，Vogel还有张）于30岁之前就做出了他们各自的重要成果是很有启发意义的。年轻常常意味着愿意在未知的方向和看似无人问津的问题上冒险——和想要获得成功的动力。对于如今这个首次获得NIH资助的年龄已经升至42岁的时代而言，这是个重要的提醒。

还值得注意的是，他们中的许多人都是在可能被一些人看作远离科学研究的常规渠道的地方做出标志性工作的（西班牙的阿利坎特；法国的国防部；丹尼斯科的公司实验室；Vilnius在立陶宛）。此外，他们的重要论文都被一流期刊拒绝了——在很久的延迟之后得以发表在并不令人瞩目的位置。这些遭遇恐怕并非偶然：这些研究地点可能给予了从事不那么热门选题研究的更大自由，但是对于如何克服期刊和评审人的怀疑与不信任态度所给予的支持帮助却是较少的。

最后，这些科学突破背后的故事鲜有灵感迸发的瞬间。它们通常是一幕群戏，演出了10年以上，在其中的演职人员共同实现了他们各自孤身奋战所无法企及的高度。对于一般公众和希冀科学生涯的青年人，这都是精彩的一课。

来源：BioArt

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