Редактирование генома и защита от его враждебного использования

Технологии редактирования генома человека («генетические ножницы») могут и возможно уже используются для целей биологического поражения различных биологических объектов, включая людей. Необходимо разработать способы международного контроля за использованием данной технологии и для обнаружения скрытого применения поражающих агентов, созданных на ее основе.

Актуальность. Технология редактирования генома активно внедряется в клиническую практику с 2021 г. для лечения наследственных болезней, в перспективе ее применение будет расширено для лечения инфекционных и соматических болезней.

Цель работы – оценить возможность ее враждебного использования.

Источниковая база исследования. Научные работы, представленные в базе данных медицинских и биологических публикаций PubMed.

Метод исследования. Аналитический.

Результаты. Системы редактирования генома позволяют осуществлять блокирование, удаление или восстановление собственных генов и фрагментов генома человека. Они могут использоваться для уничтожения экосистем, используемых человеком; разработки принципиально новых средств массового биологического поражения населения выбранных для уничтожения стран; и, даже, изменение эволюционной траектории нашего вида вплоть до его полного вымирания в течение нескольких поколений. Обращено внимание на повышенный интерес Агентства перспективных исследовательских проектов министерства обороны США (Defense Advanced Research Projects Agency, DARPA) на разработку технологий выявления и блокирования последствий редактирования генома.

Заключение. Интерес DARPA к технологиям выявления и блокирования последствий редактирования генома человека свидетельствует, что ее освоение уже перешло экспериментальный рубеж, и ее бесконтрольное использование вызывает серьезные опасения со стороны тех, кто понимает реалии современной биологической войны. Использование технологии редактирования генома должно быть урегулировано в рамках особого Протокола к Конвенции о запрещении разработки, производства и накопления запасов бактериологического (биологического) и токсинного оружия и об их уничтожении. Пока его нет, то на уровне национальных регуляторов необходимо определить границы применения продуктов, созданных на основе такой технологии, и быть готовым к их враждебному использованию.

1. Altae-Tran H, Kannan S, Suberski AJ, Mears KS, Demircioglu FE, Moeller L, et al. Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science. 2023;382(6673):eadi1910. https://doi.org/10.1126/science.adi19102

2. Marino ND, Pinilla-Redondo R, Csörgő B, Bondy-Denomy J. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nat Methods. 2020;17(5):471–79. https://doi.org/10.1038/s41592-020-0771-6

3. Knott GJ, Thornton BW, Lobba MJ, Liu JJ, Al-Shayeb B, Watters KE, et al. Broad-spectrum enzymatic inhibition of CRISPR-Cas12a. Nat Struct Mol Biol. 2019;26(4):315–21. https://doi.org/10.1038/s41594-019-0208-z

4. Maji B, Gangopadhyay SA, Lee M, Shi M, Wu P, Heler R, et al. A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9. Cell. 2019;177(4):1067–79.e19. https://doi.org/10.1016/j.cell.2019.04.009

5. Lim D, Zhou Q, Cox KJ, Law BK, Lee M, Kokkonda P, et al. A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Nat Cell Biol. 2022;24(12):1766–75. https://doi.org/10.1038/s41556-022-01005-8

6. Zhao J, Inomata R, Kato Y, Miyagishi M. Development of aptamer-based inhibitors for CRISPR/Cas system. Nucleic Acids Res. 2021;49(3):1330-44. https://doi.org/10.1093/nar/gkaa865

7. Barkau CL, O'Reilly D, Rohilla KJ, Damha MJ, Gagnon KT. Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9. Nucleic Acid Ther. 2019;29(3):136–47. https://doi.org/10.1089/nat.2018.0758

8. Camara-Wilpert S, Mayo-Muñoz D, Russel J, Fagerlund RD, Madsen JS, Fineran PC, et al. Bacteriophages suppress CRISPR-Cas immunity using RNA-based anti-CRISPRs. Nature. 2023;623(7987):601–7. https://doi.org/10.1038/s41586-023-06612-5

9. Karvelis T, Druteika G, Bigelyte G, Budre K, Zedaveinyte R, Silanskas A, et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature. 2021;599(7886):692–6 https://doi.org/10.1038/s41586-021-04058-1

10. Saito M, Xu P, Faure G, Maguire S, Kannan S, Altae-Tran H, et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. 2023;620(7974):660–8. https://doi.org/10.1038/s41586-023-06356-2.

11. Pinilla-Redondo R, Shehreen S, Marino ND, Fagerlund RD, Brown CM, Sørensen SJ, et al. Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements. Nat Commun. 2020;11(1):5652. https://doi.org/10.1038/s41467-020-19415-3

12. Madani A, Krause B, Greene ER, Subramanian S, Mohr BP, Holton JM, et al. Large language models generate functional protein sequences across diverse families. Nat Biotechnol. 2023;41(8):1099–106. https://doi.org/10.1038/s41587-022-01618-2

13. Marraffini LA. CRISPR-Cas immunity in prokaryotes. Nature. 2015;526(7571):55–61. https://doi.org/10.1038/nature15386

14. Bansal R. CRISPR-Cas: Applications in gene editing & beyond: CRISPR Cas System. 1st ed. 2023. ISBN 978-9358912173.

15. Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E. The Biology of CRISPR-Cas: Backward and Forward. Cell. 2018;172(6):1239–59. https://doi.org/10.1016/j.cell.2017.11.032

16. Xiao Y, Ng S, Nam KH, Ke A. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration. Nature. 2017;550(7674):137-41. https://doi.org/10.1038/nature24020

17. Gleditzsch D, Pausch P, Müller-Esparza H, Özcan A, Guo X, Bange G, Randau L. PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biol. 2019;16(4):504–17. https://doi.org/10.1080/15476286.2018.1504546

18. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011(6):467–77. https://doi.org/10.1038/nrmicro2577

19. Makarova KS, Koonin EV. Annotation and Classification of CRISPR-Cas Systems. Methods Mol Biol. 2015;1311:47–75. https://doi.org/10.1007/978-1-4939-2687-9_4

20. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096. https://doi.org/10.1126/science.1258096

21. Babu K, Kathiresan V, Kumari P, Newsom S, Parameshwaran HP, Chen X, et al. Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence. Biochemistry. 2021;60(49):3783–800. https://doi.org/10.1021/acs.biochem.1c00354

22. Davis AJ, Chen DJ. DNA double strand break repair via non-homologous end-joining. Transl Cancer Res. 2013;2(3):130–43. https://doi.org/10.3978/j.issn.2218-676X.2013.04.02

23. Yang H, Ren S, Yu S, Pan H, Li T, Ge S, et al. Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks. Int J Mol Sci. 2020;21(18):6461. https://doi.org/10.3390/ijms21186461

24. Song F, Stieger K. Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks. Mol Ther Nucleic Acids. 2017;7:53–60. https://doi.org/10.1016/j.omtn.2017.02.006

25. Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids. 2015;4(11):e264. https://doi.org/10.1038/mtna.2015.37

26. Tycko J, Myer VE, Hsu PD. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity. Mol Cell. 2016;63(3):355–70. https://doi.org/10.1016/j.molcel.2016.07.004

27. Cebrian-Serrano A, Davies B. CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools. Mamm Genome. 2017;28(7–8):247–61. https://doi.org/10.1007/s00335-017-9697-4

28. Hillary VE, Ceasar SA. A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/ Cas14 Proteins Utilized for Genome Engineering. Mol Biotechnol. 2023;65(3):311–25. https://doi.org/10.1007/s12033-022-00567-0

29. Saifaldeen M, Al-Ansari DE, Ramotar D, Aouida M. CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering. Cells. 202021;9(11):2518. https://doi.org/10.3390/cells9112518

30. Tadić V, Josipović G, Zoldoš V, Vojta A. CRISPR/Cas9-based epigenome editing: An overview of dCas9- based tools with special emphasis on off-target activity. Methods. 2019;164–165:109–19. https://doi.org/10.1016/j.ymeth.2019.05.003

31. Karlson CKS, Mohd-Noor SN, Nolte N, Tan BC. CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences. Plants (Basel). 2021;10(10):2055. https://doi.org/10.3390/plants10102055

32. Siksnys V, Gasiunas G. Rewiring Cas9 to Target New PAM Sequences. Mol Cell. 2016;61(6):793–4. https://doi.org/10.1016/j.molcel.2016.03.002

33. Guo M, Ren K, Zhu Y, Tang Z, Wang Y, Zhang B, et al. Structural insights into a high-fidelity variant of SpCas9. Cell Res. 2019;29(3):183–92. https://doi.org/10.1038/s41422-018-0131-6

34. Liu MS, Gong S, Yu HH, Jung K, Johnson KA, Taylor DW. Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA. Nat Commun. 2020;11(1):3576. https://doi.org/10.1038/s41467-020-17411-1

35. Rabinowitz R, Offen D. Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing. Mol Ther. 20213;29(3):937–48. https://doi.org/10.1016/j.ymthe.2020.11.009

36. Gasiunas G, Young JK, Karvelis T, Kazlauskas D, Urbaitis T, Jasnauskaite M, et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun. 2020;11(1):5512. https://doi.org/10.1038/s41467-020-19344-1

37. Behr M, Zhou J, Xu B, Zhang H. In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges. Acta Pharm Sin B. 2021;11(8):2150–71. https://doi.org/10.1016/j.apsb.2021.05.020

38. Sousa DA, Gaspar R, Ferreira CJO, Baltazar F, Rodrigues LR, Silva BFB. In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome-DNA Complexes. Pharmaceutics. 2022;14(5):1087. https://doi.org/10.3390/pharmaceutics14051087

39. Hryhorowicz M, Grześkowiak B, Mazurkiewicz N, Śledziński P, Lipiński D, Słomski R. Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles into Porcine Fibroblast. Mol Biotechnol. 2019;61(3):173–80. https://doi.org/10.1007/s12033-018-0145-9

40. Bier E. Gene drives gaining speed. Nat Rev Genet. 2022;23(1):5–22. https://doi.org/10.1038/s41576-021-00386-0

41. Vogan AA, Higgs PG. The advantages and disadvantages of horizontal gene transfer and the emergence of the first species. Biol Direct. 2011;6:1. https://doi.org/10.1186/1745-6150-6-1

42. Emamalipour M, Seidi K, Zununi Vahed S, Jahanban-Esfahlan A, Jaymand M, Majdi H, et al. Horizontal Gene Transfer: From Evolutionary Flexibility to Disease Progression. Front Cell Dev Biol. 2020;8:229. https://doi.org/10.3389/fcell.2020.00229

43. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Raguram A, Liu DR. Searchand-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149–57. https://doi.org/10.1038/s41586-019-1711-4

44. Marzec M, Brąszewska-Zalewska A, Hensel G. Prime Editing: A New Way for Genome Editing. Trends Cell Biol. 2020;30(4):257–9. https://doi.org/10.1016/j.tcb.2020.01.004

45. Li X, Zhou L, Gao BQ, Li G, Wang X, Wang Y, et al. Highly efficient prime editing by introducing samesense mutations in pegRNA or stabilizing its structure. Nat Commun. 2022;13(1):1669. https://doi.org/10.1038/s41467-022-29339-9

46. Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022;40(3):402–10 https://doi.org/10.1038/s41587-021-01039-7 Epub 2021 Oct 4. Erratum in: Nat Biotechnol. 2022;40(3):432. https://doi.org/10.1038/s41587-021-01175-0

47. Chen PJ, Liu DR. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet. 2023;24(3):161–77. https://doi.org/10.1038/s41576-022-00541-1

48. Hillary VE, Ceasar SA. Prime editing in plants and mammalian cells: Mechanism, achievements, limitations, and future prospects. Bioessays. 2022;44(9):e2200032. https://doi.org/10.1002/bies.202200032

49. Kantor A, McClements ME, MacLaren RE. CRISPR-Cas9 DNA Base-Editing and Prime-Editing. Int J Mol Sci. 2020;21(17):6240. https://doi.org/10.3390/ijms21176240

50. Huang TP, Newby GA, Liu DR. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat Protoc. 2021;16(2):1089–128. https://doi.org/10.1038/s41596-021-00525-1

51. Chen S, Jiao Y, Pan F, Guan Z, Cheng SH, Sun D. Knock-In of a Large Reporter Gene via the HighThroughput Microinjection of the CRISPR/Cas9 System. IEEE Trans Biomed Eng. 2022;69(8):2524–32. https://doi.org/10.1109/TBME.2022.3149530

52. Li Y, Li W, Li J. The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science. J Genet Genomics. 2021;48(8):661–70. https://doi.org/10.1016/j.jgg.2021.05.001

53. Molla KA, Sretenovic S, Bansal KC, Qi Y. Precise plant genome editing using base editors and prime editors. Nat Plants. 2021;7(9):1166–87. https://doi.org/10.1038/s41477-021-00991-1

54. Gilmore TD. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene. 2006;25(51):6680–4. https://doi.org/10.1038/sj.onc.1209954

55. Böhm S, Splith V, Riedmayr LM, Rötzer RD, Gasparoni G, Nordström KJV, et al. A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation. Sci Adv. 2020;6(34):eaba5614. https://doi.org/10.1126/sciadv.aba5614

56. Riedmayr LM, Hinrichsmeyer KS, Karguth N, Böhm S, Splith V, Michalakis S, et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat Protoc. 2022;17(3):781–18. https://doi.org/10.1038/s41596-021-00666-3

57. Omachi K, Miner JH. Comparative analysis of dCas9-VP64 variants and multiplexed guide RNAs mediating CRISPR activation. PLoS One. 2022;17(6):e0270008. https://doi.org/10.1371/journal.pone.0270008

58. Hunt C, Hartford SA, White D, Pefanis E, Hanna T, Herman C, et al. Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice. Nat Commun. 2021;12(1):2770. https://doi.org/10.1038/s41467-021-22932-4

59. Mittal N, Guimaraes JC, Gross T, Schmidt A, Vina-Vilaseca A, Nedialkova DD, et al. The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan. Nat Commun. 2017;8(1):457. https://doi.org/10.1038/s41467-017-00539-y

60. Heckl D, Charpentier E. Toward Whole-Transcriptome Editing with CRISPR-Cas9. Mol Cell. 2015;58(4):560-2. https://doi.org/10.1016/j.molcel.2015.05.016

61. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015;517(7536):583–8. https://doi.org/10.1038/nature14136

62. Montefiori LE, Nobrega MA. Gene therapy for pathologic gene expression. Science. 2019;363(6424):231–2. https://doi.org/10.1126/science.aaw0635

63. Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18(5):358–78. https://doi.org/10.1038/s41573-019-0012-9

64. Alerasool N, Segal D, Lee H, Taipale M. An efficient KRAB domain for CRISPRi applications in human cells. Nat Methods. 2020;17(11):1093–96. https://doi.org/10.1038/s41592-020-0966-x

65. Stoll GA, Pandiloski N, Douse CH, Modis Y. Structure and functional mapping of the KRAB-KAP1 repressor complex. EMBO J. 2022;41(24):e111179. https://doi.org/10.15252/embj.2022111179

66. Zhang X, Blumenthal RM, Cheng X. Keep Fingers on the CpG Islands. Epigenomes. 2024;8(2):23. https://doi.org/10.3390/epigenomes8020023

67. Ueda J, Yamazaki T, Funakoshi H. Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges. Int J Mol Sci. 2023;24(5):4778. https://doi.org/10.3390/ijms24054778

68. Kuscu C, Mammadov R, Czikora A, Unlu H, Tufan T, Fischer NL, et asl. Temporal and Spatial Epigenome Editing Allows Precise Gene Regulation in Mammalian Cells. J Mol Biol. 2019;431(1):111–21. https://doi.org/10.1016/j.jmb.2018.08.001

69. Cai R, Lv R, Shi X, Yang G, Jin J. CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation. Int J Mol Sci. 2023;24(19):14865. https://doi.org/10.3390/ijms241914865

70. Barkau CL, O'Reilly D, Eddington SB, Damha MJ, Gagnon KT. Small nucleic acids and the path to the clinic for anti-CRISPR. Biochem Pharmacol. 2021;189:114492. https://doi.org/10.1016/j.bcp.2021.114492

71. Pawluk A, Bondy-Denomy J, Cheung VH, Maxwell KL, Davidson AR. A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. mBio. 2014;5(2):e00896. https://doi.org/10.1128/mBio.00896-14

72. Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR. Bacteriophage genes that inactivate the CRISPR/ Cas bacterial immune system. Nature. 2013;493(7432):429–32. https://doi.org/10.1038/nature11723

73. Seamon KJ, Light YK, Saada EA, Schoeniger JS, Harmon B. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity. Anal Chem. 2018;90(11):6913–21. https://doi.org/10.1021/acs.analchem.8b01155

74. Jin L, Wang W, Fang G. Targeting protein-protein interaction by small molecules. Annu Rev Pharmacol Toxicol. 2014;54:435–56. https://doi.org/10.1146/annurev-pharmtox-011613-140028

75. Maji B, Gangopadhyay SA, Lee M, Shi M, Wu P, Heler R, et al. A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9. Cell. 2019;177(4):1067–79.e19. https://doi.org/10.1016/j.cell.2019.04.009

76. O'Reilly D, Kartje ZJ, Ageely EA, Malek-Adamian E, Habibian M, Schofield A, et al. Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity. Nucleic Acids Res. 2019;47(2):546–58. https://doi.org/10.1093/nar/gky1214

77. Kartje ZJ, Barkau CL, Rohilla KJ, Ageely EA, Gagnon KT. Chimeric Guides Probe and Enhance Cas9 Biochemical Activity. Biochemistry. 2018;57(21):3027-31. https://doi.org/10.1021/acs.biochem.8b00107

78. Barkau CL, O'Reilly D, Rohilla KJ, Damha MJ, Gagnon KT. Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9. Nucleic Acid Ther. 2019;29(3):136–47. https://doi.org/10.1089/nat.2018.0758

79. Zeng C, Zhang C, Walker PG, Dong Y. Formulation and Delivery Technologies for mRNA Vaccines. Curr Top Microbiol Immunol. 2022;440:71–110. https://doi.org/10.1007/82_2020_217

80. Qiu M, Li Y, Bloomer H, Xu Q. Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing. Acc Chem Res. 2021;54(21):4001–11. https://doi.org/10.1021/acs.accounts.1c00500

81. Kim J, Eygeris Y, Gupta M, Sahay G. Self-assembled mRNA vaccines. Adv Drug Deliv Rev. 2021;170:83–112. https://doi.org/10.1016/j.addr.2020.12.014

82. Wang X, Liu S, Sun Y, Yu X, Lee SM, Cheng Q, et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat Protoc. 2023;18(1):265–91. https://doi.org/10.1038/s41596-022-00755-x

83. Parhiz H, Atochina-Vasserman EN, Weissman D. mRNA-based therapeutics: looking beyond COVID-19 vaccines. Lancet. 2024;403(10432):1192–204. https://doi.org/10.1016/S0140-6736(23)02444-3

84. Iqbal Z, Rehman K, Mahmood A, Shabbir M, Liang Y, Duan L, Zeng H. Exosome for mRNA delivery: strategies and therapeutic applications. J Nanobiotechnology. 2024;22(1):395. https://doi.org/10.1186/s12951-024-02634-x

85. DiEuliis D, Giordano JJ. Safely balancing a double-edged blade: identifying and mitigating emerging biosecurity risks in precision medicine. Front Med (Lausanne). 2024;11:1364703. https://doi.org/10.3389/fmed.2024.1364703

86. DiEuliis D, Giordano J. Why Gene Editors Like CRISPR/Cas May Be a Game-Changer for Neuroweapons. Health Secur. 2017;15(3):296–302. https://doi.org/10.1089/hs.2016.0120

87. Ajitkumar A, De Jesus O. Huntington Disease. 2023 Aug 23. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. PMID: 32644592

88. Shin JW, Hong EP, Park SS, Choi DE, Seong IS, Whittaker MN, Kleinstiver BP, Chen RZ, Lee JM. Allelespecific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9. JCI Insight. 2022;7(19):e141042. https://doi.org/10.1172/jci.insight.141042

89. Cheng Y, Zhang S, Shang H. Latest advances on new promising molecular-based therapeutic approaches for Huntington's disease. J Transl Int Med. 2024;12(2):134–47. https://doi.org/10.2478/jtim-2023-0142

90. Sahel DK, Giriprasad G, Jatyan R, Guha S, Korde A, Mittal A, Bhand S, Chitkara D. Next-generation CRISPR/Cas-based ultrasensitive diagnostic tools: current progress and prospects. RSC Adv. 2024;14(44):32411–35. https://doi.org/10.1039/d4ra04838e

91. Wong C. UK first to approve CRISPR treatment for diseases: what you need to know. Nature. 2023;623(7988):676–77. https://doi.org/10.1038/d41586-023-03590-6

92. Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies. Cell. 2024;187(5):1076–100. https://doi.org/10.1016/j.cell.2024.01.042