Soroka D. S., Sokolova I. Y., Gavrilyuk V. G., Sklyar T. V

LITERATURE REVIEWS

Scentific article

Today CRISPR / Cas9 technology is considered a significant breakthrough in biology, since it allows highprecision, cost-effective and quick editing, knock-out and cutting of genes and even genes. For the first time, the CRISPR locus, which is a repeat genetic element separated by variable spacers, was invented in the E. coli genome in 1987 by a group of Japanese scientists led by Yoshizuki Isino at the University of Osaka (Journal of bacteriology, 1989). In 1993, the same genetic elements were discovered by the Spanish researcher F. Mojica in the genome of the Archaea Haloferax mediterranei and named their DNA Clustered Regularly Interspaced Short Palindromic Repeats. Until 2000, Mojica found their presence in 20 microorganisms, including Yersinia pestis. It has been found that the CRISPR locus is part of an unknown mechanism that aims to protect against foreign DNA and is an expression of the adaptive immunity of microorganisms against viruses. Typical CRISPR locus consist of several direct replays separated by spacers – regions of variable DNA sequences. The CRISPR repeat contains 23-47 pairs of bases, and the spacer is 21-72 pairs. The number of “repeat / spacer” groups can reach 375, but often less than 50. The bacteria usually contain several CRISPR loci. Each locus provides protection against a particular virus. In the archaea, Methanocaldococcus jannaschii identified 18 loci (1% genome). CRISPRs are in the chromosome or in the plasmid DNA. In 2002, the operon with cas-genes in CRISPR locus were also discovered. They encode the synthesis of Casproteins – enzymes involved in the splitting of foreign DNA of viruses. Each Cas-protein carries several domains: typical nucleases, helicase, polymerases, DNA-binding proteins. This Cas-proteins are polyfunctional and capable of performing all of these enzymes. The Cas9 protein isolated from Streptococcus pyogenes cells consists of 5 domains (REC I, REC II, HNH, RuvC, RAM) and the additional Bridge Helix. Rec I is responsible for binding RNA Guide, REC II has not yet been sufficiently studied; Bridge Helix initiates DNA splitting when bound to a target DNA. The PAM-interacting domain provides the specificity of PAM and is responsible for binding to a specific DNA target. HNH and RuvC are endonucleases domains that cut the DNA. An important role in the recognition of foreign DNA by the CRISPR system is played by the RNA Guide. It, due to complementarity with the Target DNA, forms a duplex, which is recognized by the Cas9 protein and cleaved by its endonuclease activity. Cas9 allows the cleavage of any nucleotide sequence. The selectivity of Cas9 is a consequence of the complementarity of the RNA guide and DNA target. The mechanism for the formation of adaptive immunity under the action of CRISPR/Cas-system in invasion of viral or plasmid DNA into S. aureus cell involves two main stages – A and B. At stage A (Immunization) Cas2 is activated and splits the foreign DNA. A new spacer is created – a fragment of viral DNA inserted in the CRISPR cassette. When a virus hits the bacterium again (stage B – Immunity), transcription is carried out on the matrix of CRISPR cassettes with formation of mRNA, which carries information about all cassette spacers. As a result of processing under action of Cas2 mRNA is hydrolyzed with formation of individual RNA molecules, among which there is already a RNA-guide to detect the virus that penetrated the cell. With help of a specific RNA guide, Cas1 targets the viral DNA, cause it degradation. The CRISPR/Cas not only provides adaptive immunity against alien genetic elements, but also acts as a limiter for the horizontal transfer of viruses, plasmids, transposons between different bacterial cells. CRISPR/Cas methods are used in genetic engineering of eukaryotes and prokaryotes. Thus a new variety of rice was obtained, whose sprouts look like dwarf albinos. The possibilities of introducing CRISPR/Cas systems into cultivated plants for the creation of antiviral immunity are explored. Also, the attempts to edit genomes of pigs, cattle, monkeys, mice, bees. The results of the experiment, in which 62 endogenous retroviruses were inactivated with the help of CRISPR/Cas technology in the pig genome, were published. Thanks to these results, in the future, xenotransplantation of organs from pig to person will be possible. Methods based on CRISPR/Cas9 can also be used in medicine for treatment of various diseases: viral, immune, oncological, cardiovascular, hereditary, including Down’s syndrome, sickle cell anemia, pigment retinitis, β-thalassemia and others. In 2013 there was a publication about editing an abnormal gene in stem cells of a patient with cystic fibrosis. In 2015, Chinese scientists led by Junjiu Huang have used CRISPR technology to modify human embryonic genes. The ability to edit the genome also causes various ethical issues that need to be carefully considered.

CRISPR/Cas9, edition of genome, immunity of bacteria against viruses, RNA guide, Cas-genes, Cas9.

1.  Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology. 1987;169(12):5429-33. DOI: 0021- 9193/87/125429-05$02.00/0
2. Lander ES. The Heroes of CRISPR. Cell. 2016;164(1-2):18-28. DOI: 10.1016/j.cell.2015.12.041
3.  Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution. 2005;60(2):174-82. DOI: 10.1007/s00239-004-0046-3
4. Datsenko K, Pougach K, Tikhonov A. Molecular memory of prior infections activates the CRISPR. Cas adaptive bacterial immunity system. Nat. Commun. 2012;3:945. DOI: 10.1038/ncomms1937
5.  Ki Hyun Nam, Fran Ding, Charles Haitjema, Qingqiu Huang, Matthew PDe Lisa, Ailong Ke. Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) – аassociated Cas2 Protein. J. Biol. Chem. 2012;287(43):35943- 52. DOI: 10.1074/jbc.M112.382598
6. Horvath Р, Barrangou R. CRISPR/Cas, the Immune System of Bacteria and Archaea. Science. 2010;327(5962):167-70. DOI: 10.1126/science.1179555
7.  Jinek M, Chyilinski K, Fonfara I, Hauer M. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-21. DOI: 10.1126/science.1225829
8. Barrangou R. Diversity of CRISPR-Cas immune systems and molecular machines. Genome Biology. 2015;16:247-57. DOI: 10.1186/s13059- 015-0816-9
9. Makarova KS, Koonin EV. Annotation and Classification of CRISPR-Cas Systems. Methods in Molecular Biology. 2015;1311:47-75. DOI: 10.1007/978-1-4939-2687-9_4
10.  Makarova S. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011;9(6):467-77. DOI: 10.1038/nrmicro2577
11. Jinek M, Jiang F, Taylor DW. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014;343(6176):1247997. DOI: 10.1126/science.1247997
12. Nishimasu H, Ran FA, Hsu PD. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5):935-49. DOI: 10.1016/j. cell.2014.02.001
13.  Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Саs9. Nature. 2014;507(7490):62-7. DOI: 10.1038/nature13011
14.  Anders C. Niewoehner O, Duerst A, Martin J. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. 2014;513(7519):569-73. DOI: 10.1038/nature13579
15. Hiroshi Nishimasu, Le Cong, Winston XYan, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, et al. Crystal Structure of Staphylococcus aureus Cas9. Cell. 2015;162(5):1113-26. DOI: 10.1016/j.cell.2015.08.007
16. Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. 2010;468(7320):67-71. DOI: 10.1038/nature09523
17. Erik J. Sontheimer and Luciano A. Marraffini. MICROBIOLOGY: Slicer for DNA. Nature. 2010;468(7320):45-6. DOI: 10.1038/468045a
18. Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature. 2013;497(7448):254-7. DOI: 10.1038/nature12048
19. Nemudryiy AA, Valetdinova KR, Medvedev SP, Zakiyan SM. Sistemyi redaktirovaniya genomov TALEN i CRISPR/Cas – instrumentyi otkryitiy. Acta naturae. 2014;3(22):20-42. [in Russian].
20. Mali Prashant, Esvelt Kevin M, Church George M. Cas9 as a versatile tool for engineering biology. Nature Methods. 2013;10(10):957-63. DOI: 10.1038/nmeth.2649
21. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 2014;32(12):1262-7. DOI: 10.1038/nbt.3026
22. Mojica F, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009;155(3):733-40. DOI: 10.1099/mic.0.023960-0
23.  Elizabeth Pennisi. The CRISPR Craze. Science. 2013;341(6148):833-6. DOI: 10.1126/science.341.6148.833
24. Zaidi SS, Tashkandi M, Mansoor S, Mahfouz MM. Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance. Frontiers in plant science. 2016;7:1673. DOI: 10.3389/fpls.2016.01673
25.  Wang Zhongde. Genome engineering in cattle: recent technological advancements. Chromosome Research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 2015;23(1):17-29. DOI: 10.1007/s10577-014-9452-6
26. Niemann H, Petersen B. The production of multi-transgenic pigs: update and perspectives for xenotransplantation. Transgenic Research. 2016;25(3):361-74. DOI: 10.1007/s11248-016-9934-8
27. Kohno H, Suenami S, Takeuchi H, Sasaki T, Kubo T. Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L. Zoological science. 2016;33(5):505-12. DOI: 10.2108/zs160043
28.  Yang Luhan, Güell M, Niu Dong, George H, Lesha E, Grishin D, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350(6264):1101-4. DOI: 10.1126/science.aadll91
29.  Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/ Cas-mediated genome engineering. Cell. 2013;154(6):1370-9. DOI: 10.1016/j.cell.2013.08.022
30. Goodman MA, Moradi Manesh D, Malik P, Rothenberg ME. CRISPR/Cas9 in allergic and immunologic diseases. Expert review of clinical immunology. 2016;13(1):5-9. DOI: 10.1080/174666X.2017.1241711
31. Borges TJ, Murakami N, Riella LV. Current status of alloimmunity. Current opinion in nephrology and hypertension. 2016;25(6):556-62. DOI: 10.1097/MNH.0000000000000267
32.  Yin Hao, Xue Wen, Chen Sidi, Bogorad RL, Benedetti E, Grompe M, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology. 2014;32(6):551-3. DOI: 10.1038/nbt.2884
33. Yang Y, Zhang X, Yi L, Hou Z, Chen J, Kou X, et al. Naive induced pluripotent stem cells generated from β-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. Stem cells translational medicine. 2016;5(1):8-19. DOI: 10.5966/sctm.2015-0157
34. Schwank G, Koo Bon-Kyoung, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 2013;13(6):653-8. DOI: 10.1016/j.stem.2013.11.002
35. Jakočiūnas T, Jensen MK, Keasling JD. CRISPR/Cas9 advances engineering of microbial cell factories. Metabolic Engineering. 2016;34:44-59. DOI: 10.1016/j.ymben.2015.12.003

«Bulletin of problems biology and medicine» Issue 1 Part 2 (149), 2019 year, 46-51 pages, index UDK 577.21: 579.25: 571.27

10.29254/2077-4214-2019-1-2-149-46-51