CRISPR-epic and its heroes

Olga Volkova, "Biomolecule"

A lot has been said about prokaryotic immune systems called CRISPR-Cas, including on the "biomolecule", but the details of their discovery and study usually remained in the shadows. We decided to fill this gap slightly by referring mainly to Eric Lander's recent investigation, which covered a 20-year biography of CRISPR – "the history of ideas and the history of people."

This story began in the Spanish port town of Santa Pola, where, according to Eric Lander (Fig. 1), the beautiful coast and extensive salt marshes have attracted vacationers, flamingos and salt producers for centuries [1].

Figure 1. Eric Lander (Eric Steven Lander). Mathematician, geneticist, one of the leaders of the Human Genome project, director of the Broad Institute of MIT and Harvard, co-chairman of the Council on Science and Technology under the President of the United States. Photos from the website news.cs.washington.edu .

And in the salt there, unnoticed by vacationers, extreme athletes from the world of archaea were basking. In 1989, Francisco Mojica, a young doctoral student at Aliquonte University, became interested in their genetic stuffing (Fig. 2). In the genome of Haloferax mediterranei, he found groups of almost perfect and almost palindromic straight 30-nucleotide repeats separated by spacers – unique, non-repeating sections of approximately the same length. Mohika compared these strange clusters with similar (but not similar in sequence) repeats in the genome of Escherichia coli, noticed by the Japanese in 1987. However, for scientists from Osaka, repetitions have remained a simple side find.

In 1995, Mojica published an article on the discovery of a new class of prokaryotic repeats called short regularly spaced repeats (SRSR). Later, at his suggestion, they were renamed clustered regularly interspaced short palindromic repeats (CRISPR).

By 2000, the same Mojica revealed the same repeats in a mass of archaea and bacteria, including pathogens of dangerous infections – Mycobacterium tuberculosis and Yersinia pestis.

In 2002, a research group from the Netherlands noticed that a similar group of genes always adjoins CRISPR. For which these genes were named cas (CRISPR-associated genes) [2]. Of course, everyone was thinking about the functions of this system: after all, since it is so common in prokaryotes, it means that for some reason they really need it.

And so, in 2003, Mohika decided to "sweeten" spacers – to look for similar fragments in the world database of DNA sequences. Again. He had also tried before, but without success: the base was still weak. And now we were lucky: there was a match of one of the CRISPR spacers of the E. coli strain resistant to phage P1 with the DNA of this very phage. By the end of the week, 88 spacers had already found "relatives", and the majority were among phages and conjugative plasmids. Then Mojica suggested that CRISPR encode instructions for adaptive (acquired) immunity reactions that protect prokaryotes from infections.

After drinking cognac with colleagues in honor of the birth of the idea, Mohika started writing an article. Where to? Of course, in Nature! In view of the extreme importance of the discovery – so it seemed to him. Lander called the next 18 months of the Spaniard's life an odyssey of disappointments. The editors of Nature rejected the article, for some unknown reason deciding that the idea of Mojica is not new at all; in the same way, but doubting its importance, in 2004, PNAS responded, and then from Molecular Microbiology and Nucleic Acid Research. The editors of the Journal of Molecular Evolution turned out to be more prescient, and after a year (!) The completely exhausted Mohika saw his hypothesis about the biological role of CRISPR outlined on quite decent paper. Not Nature, but still... 10 years have passed, and, as one of the leading CRISPR researchers Konstantin Severinov noted (Fig. 3), Nature is now ready to print works even at the student coursework level if they relate to CRISPR-Cas [3].

Figure 2. Francisco Juan Martínez Mojica and Gilles Vergnaud. Photos from websites economia3.com and ensta-paristech.fr .

It is noteworthy that at the same time, in 2004-2005, two more scientific groups experienced a similar odyssey, trying to publish their ideas about the functions of CRISPR. These groups approached the topic of CRISPR from completely different sides. Christine Porcel and Gilles Verneuw (Fig. 2) worked on the instructions of the French Ministry of Defense, typing strains of plague bacillus by DNA repeats - that is, selecting genetic markers capable of distinguishing Y.pestis isolates of different origin. The group showed that closely related strains can sometimes differ only by CRISPR loci and that new spacers homologous to phage DNA are embedded at the same end of the locus. Four journals refused to accept an article with the idea that CRISPR is a bacterial memory of past genetic aggressors. The same thing, but with regard to streptococcal CRISPR, was claimed by Alexander Bolotin of the French National Institute of Agricultural Research. And he was the first to suggest exactly how this immune system works. Bolotin's hypothesis, although not entirely correct regarding the mechanism, found "its" magazine on just the second attempt.

Figure 3. Konstantin Severinov, Virginijus Šikšnys and Feng Zhang. Photos from websites the-village.ru , qswownews.com and twitter.com accordingly.

I must say that the study and classification of Cas proteins (only they were not called that then) Since the late 1990s, the group of Evgeny Kunin (Fig. 4), an NCBI expert in the field of bioinformatics and evolutionary biology, has been engaged. Kira Makarova and other members of the group published an article in 2002 about the possible participation of these proteins in DNA repair (which, by the way, also happens [4]). In the same year, the Dutch linked them topologically with CRISPR cassettes and called them Cas. Everything fell into place, and already in 2005, Kunin's group, like Mohika and Vernya, calculated the immune purpose of the CRISPR-Cas system. And, like Bolotin, suggested RNA interference as an immune mechanism. A hypothetical scheme of prokaryotic autovaccination for the benefit of offspring – with the transfer of acquired immunity in the spirit of Lamarckism – took shape in an article in 2006 [5].

The adaptive immune system of prokaryotes was shown in action by industrial microbiologists, called upon by the transnational food industry to genotype strains of Streptococcus thermophilus: for the manufacture of yoghurts and cheeses, it was necessary to learn how to select phage–resistant "workhorses", and even better - to make them so. Philip Horvath's group (Fig. 4) linked this resistance to CRISPR shortly before the publication of Mohiki's article, and by 2006 purposefully derived phage-resistant strains, noting that streptococcus confidence in phage resistance grew in proportion to the number of spacers borrowed from these viruses. But even the single-nucleotide differences between the spacer and its complementary phage target were enough to disable the protection.

The same group of microbiologists found out the functions of Cas7 and Cas9 proteins (the future hero of genetically engineered thrillers was then called Cas5 or Csn1) in adaptive bacterial immunity. In 2010, the team already knew exactly how and where the Cas9 target, the type II CRISPR killer protein, cuts. And that for its operation, the target must necessarily contain three specific nucleotides – the PAM sequence (proto-spacer adjacent motif). All this not only advanced scientists in understanding the work of the system, but also increased the efficiency of the production of starter cultures, and consequently, the volume of their sales. And now there is a huge probability that there is a "crispized" yogurt in our refrigerator. The manufacturer assures the particularly timid that there are no "GMOs" – artificially modified streptococci – and there are only those that have accumulated spacers naturally, being attacked by phages under the strict control of microbiologists [6].

The device of the CRISPR-Cas system and its operation - as it is known now – have been analyzed more than once on the "biomolecule". We offer, for example, to view the latest competitive infographic "Just about the difficult: CRISPR/Cas" [4].

Figure 4. Eugene V. Koonin, Philippe Horvath and John van der Oost. Photos from websites proteome.nih.gov , copainsdavant.linternaute.com and cnb.csic.es accordingly.

The Dutchman John van der Oost (Fig. 4), who became infected with interest in CRISPR from Kunin, together with colleagues biochemically described the Cas proteins of the Cascade complex (type I CRISPR system) and their participation in the cutting of working transcripts of the CRISPR cassette (sgRNA), and in 2008 for the first time experimentally "programmed" CRISPR to the destruction of a specific phage "inoculated" E. coli from phage λ, indirectly showing that the target of sgRNA is DNA, not RNA. In the same year, this was confirmed by Marraffini and Sontheimer (Fig. 5), who suggested that CRISPR is a programmable nuclease system that can be used to dissect the necessary DNA sections in vitro and, possibly, even in eukaryotic cells.

Figure 5. Luciano Marraffini and Erik Sontheimer. Photos from websites diariohoy.net and umassmed.edu .

In 2010-2011, infectious microbiologists Emmanuel Charpentier and Joerg Vogel (Fig. 6), studying transcriptomes (a set of RNA) of pathogens, including Streptococcus pyogenes, discovered a missing link in the type II CRISPR system - a partner of sgRNA, which was called tracrRNA (trans-activating CRISPR RNA). They also showed that this molecule, complementary to CRISPR repeats and encoded in the immediate vicinity of the locus, is needed for processing ("maturation") sgRNA. In 2012, Cas9's assistance in destroying the target was added to this function.

Back in 2007, Lithuanian biochemist Virginijus Shikshnis (Fig. 3) preferred to participate in the growing CRISPR epic to the tedious study of the structure of nucleases. In 2011, his team managed to transfer the type II CRISPR system from streptococcus to E. coli intact and functionally intact. It became obvious that for targeted target cutting, only three components are really needed: sgRNA–tracrRNA–Cas9, which are quite workable even in heterologous systems.

And then two groups at once (one – Shikshnis, the other – Emmanuel Charpentier and Jennifer Dudny (Fig. 6), an RNA expert from the University of California) practiced in vitro obtaining functional systems with a certain specificity. And in 2012, both groups reported on the potential of the simplest CRISPR-Cas9 system for RNA-programmed genome editing. At the same time, Charpentier and Dudna also successfully combined sgRNA and tracrRNA into one shortened molecule – sdRNA. The only guiding RNA subsequently made life much easier for genetic engineers.

Figure 6. Jörg Vogel, Emmanuelle Charpentier and Jennifer Doudna. Photos from websites ukw.de , news.embl.de and ru.pinterest.com accordingly.

For example, such as Feng Zhang (Fig. 3). From the age of 16, immersed in the wilds of molecular biology, and then optogenetics, he dreamed of eradicating neuropsychiatric ailments by controlling the activity of neural genes with the help of light. And he succeeded in something. However, in 2011, he heard about CRISPR, and in mid-2012, Zhang already had a ready-made three-component editing system optimized for mammalian cells and consisting of streptococcal Cas9, tracrRNA and CRISPR locus, "aimed" at 16 sites of human and mouse genomes at once. The system worked with decent accuracy and efficiency: mutations were successful – both deletions and substitutions of DNA fragments. After the publication of the article by Charpentier and Dudna, Zhang switched to a two-component system, but found out that in vivo the full-size version of the sdRNA works more efficiently, rather than a shortened one. Zhang's article, published in January 2013 in Science, became the most cited in this field, and his CRISPR-Cas9 coding vectors have been flying like hot cakes for three years through the non-profit organization Addgene [1]. Vrochem, as well as reagents of other laboratories.

Almost simultaneously, an article was published by the pupils of George Church, known for his provocative, but very tempting, ideas. Prashant Mali and Luhan Yang (Figure 7) performed the same CRISPR-Cas9 enhancements as Zhang and reported successful genome editing of human cells. At the same time, they evaluated the advantages of full-size sdRNA in vivo earlier than Zhang's team.

However, news about the successful operation of this programmable nuclease system in vivo spread throughout the scientific community even before the release of landmark publications. And in 2013, a real CRISPR epidemic began: the Google search engine was sick of queries with the word "CRISPR", and biologists around the world cut and cut the genomes of yeast, plants, worms, flies, mice, fish, axolotls and monkeys.

Figure 7. Luhan Yang and George Church, dreaming of the resurrection of mammoths. Photos from websites forbes.com and deutschlandfunk.de .

In 2015, the teams of Zhang, van der Ost and Kunin described the work of a new, "minimal" type of natural CRISPR systems - CRISPR–Cpf1 (type V-A). Before that, the system was considered hypothetical: it was discovered by a special bioinformatic program in Francisella novicida and a number of other bacteria. It turned out that CRISPR-Cpf1 does not need tracrRNA. An equally important "highlight" of the system is the effector protein Cpf1, which can also work in human cells: it recognizes a different type of PAM in the target and cuts the target differently than Cas9, leaving "sticky" ends (the incisions of DNA chains are located not opposite each other, but with an offset) [7]. The same protein, as Charpentier's group reported a year later, is actively involved in the maturation of sgRNA [8].

In 2016, the journal Science published the results of studying another, also two-component, type VI CRISPR system – with the effector protein C2c2, attacking not DNA, but exclusively RNA. Among the authors are Severinov, Kunin, Zhang and Lander himself [9]. Scientists expressed the system from Leptotrichia shahii in E. coli and showed that C2c2 is a ribonuclease directed by SRNA, cutting, for example, phage RNA, but not directly at the recognition site, and not only it. After activation, that is, interaction with the target, C2c2 arranges a real massacre – destroys any RNA, bringing the infected bacterium to suicide. Actually, human cells also fight viruses in a similar way. Since the CRISPR-C2c2 system operates on the principle of RNA interference, it seems that scientists who previously assumed exactly such a mechanism for the implementation of bacterial immunity were not very wrong.

This was the CRISPR theory of 2015-2016. Meanwhile, CRISPR practice was at the peak of not only scientific, but also public attention. In 2015, experiments with non-viable human embryos were detonated [10, 11]. Despite the mind-boggling therapeutic prospects, the shadow of a moratorium hangs over the use of CRISPR technology in relation to human germ and embryonic cells. 

This and other moratoriums in biology are mentioned in the competitive article "Once again about GMOs" [12], dedicated to the issues of genetic modification and interesting applications of GMOs

And if the problem of "designer children" excites the minds of ordinary people, then scientists are mainly concerned about the imperfection of technology. But even the most cautious admit that the process cannot be stopped. And not only in the outpost of adventurous biotechnology, China. It is said that work on the correction of the germ line can be allowed in California [3].

The latest experiment of Chinese scientists on CRISPR therapy of lung cancer is described in the material "From words to deeds: CRISPR-Cas technology was first used for the treatment of oncological diseases" [13]

At the end of his historical investigation, Lander draws interesting conclusions [1].

1. "If you only knew what kind of trash..." You never know what a major medical breakthrough will come out of. The first heroes of the CRISPR epic "designer children" never dreamed of: someone solved the riddle of bizarre prokaryotic DNA repeats, someone was recruited for work related to bioweapons, someone - to increase the efficiency of dairy production.
2. There are many young people among the CRISPR heroes, even under 30 years of age.
3. Many participants in the story made their key findings in places that someone - for example, the editor of an authoritative magazine – could call "the backyards of science", and this could contribute to the "odyssey of disappointments".
4. Discoveries based on hypotheses and made using classical, "wet" methods are increasingly crowded with discoveries based on big data and made in a "dry" way – bioinformatically [14, 15].
5. Scientific breakthroughs are less and less a "eureka". Usually this is the fruit of many years of collective efforts, where the contributions of the participants are not just summed up, but give rise to something more in synergy.

Literary and patent fights

The research of CRISPR systems, according to the participants of the described story, took place in an atmosphere of close cooperation and goodwill. However, awareness of the scope and prospects for the use of CRISPR-Cas9 technology led to the outbreak of the largest patent dispute. Dudna and Charpentier, together with colleagues, applied for a patent six months earlier than Zhang, but his application was considered in a special, accelerated mode. Therefore, in 2014, Zhang became the owner of a patent for CRISPR-Cas9 technology (regarding its use in eukaryotic cells). Of course, this did not suit the group that applied earlier. And off it went... Now the patent commission will have to read into articles, laboratory journals, correspondence, etc. and conclude about the priority of one of the parties in creating the technology (again, taking into account the objects of its application). It is not yet known how the dispute will end, but not only developers, but also directly or indirectly related biotech companies will be affected or won.

All this leads to the fact that after the publication of Lander's article "The Heroes of CRISPR" [1], a heated debate (sometimes called shitstorm) broke out about the goals and appropriateness of its release in the midst of a patent battle. The "camp" of Charpentier and Dudna accused Lander of skillfully using literary means to belittle the merits of these two scientists and "bulge" Zhang's contribution, since Lander heads the institute where he works. The charges are very controversial, and people who are not very privy to the backstage details, when reading this lively and rich article, are unlikely to get such an impression. In addition, the editors of Cell did not consider it necessary to mention a conflict of interest, since it was not about the obvious financial benefit of the author. Charpentier and Dudna themselves were dissatisfied with the coverage of their work. On the other hand, writing such stories is often a thankless task, unless portraits of heroes are already hung on the walls of school classrooms. A rare living and living hero remains satisfied with the number of lines allocated to him: he himself knows and wants to say much more...

Surprisingly, George Church, on the contrary, complained about the too frequent mention of his name to the detriment of young people, on whom, in fact, all the experiments were based. And Lander agreed with this, noting, however, that it was simply unrealistic to mention all the co-authors on several pages, and sometimes the choice was forced to be made in favor of the team leader (Lander's corrective remarks are posted in the comments to his article on the Cell website). And in the summer, Nature magazine took over the baton, deciding to pay tribute to the "unsung" heroes, those who have always been hidden by the shadow of famous chefs. The author of the article was primarily interested in how the breakthrough results obtained during the study/development of CRISPR systems affected the future fate of the "CRISPR youth" [16].

Literature

1. Lander E.S. (2016). The Heroes of CRISPR. Cell. 164 (1–2), 18–28;
2. Jansen R., Embden J.D., Gaastra W., Schouls L.M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43 (6), 1565–1575;
3. Ershov A. (2016). "It's just very beautiful." Konstantin Severinov on a new type of CRISPR systems and the latest trends in genome editing. Site N+1;
4. Biomolecule: "Just about complicated: CRISPR/Cas";
5. Makarova K.S., Grishin N.V., Shabalina S.A., Wolf Y.I., Koonin E.V. (2006). 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. Biol. Direct. 1, 7;
6. Grens K. (2015). There’s CRISPR in your yogurt. The Scientist;
7. Zetsche B., Gootenberg J.S., Abudayyeh O.O., Slaymaker I.M., Makarova K.S., Essletzbichler P. et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163 (3), 759–771;
8. Fonfara I., Richter H., Bratovič M., Le Rhun A., Charpentier E. (2016). The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 532 (7600), 517–521;
9. Abudayyeh O.O., Gootenberg J.S., Konermann S., Joung J., Slaymaker I.M., Cox D.B. et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 353 (6299), aaf5573;
10. Liang P., Xu Y., Zhang X., Ding C., Huang R., Zhang Z. et al. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 6 (5), 363–372;
11. Biomolecule: "Mutagenic chain reaction: genome editing on the verge of fiction";
12. biomolecule: "Once again about GMOs";
13. Biomolecule: "From words to deeds: CRISPR-Cas technology was used for the first time for the treatment of oncological diseases";
14. biomolecule: "The computational future of biology";
15. Biomolecule: "The Philip Haitovich Research Group, or how biologists work with large amounts of data";
16. Ledford H. (2016). The unsung heroes of CRISPR. Nature. 535 (7612), 342–344.

Portal "Eternal youth" http://vechnayamolodost.ru  30.11.2016