CRISPR-CAS UYGULAMALARI, POTANSİYEL RİSKLER VE YASAL DÜZENLEMELER

Öz

CRISPR-Cas teknolojisi, canlı bir organizmanın genomunu, endojen genlerin modifikasyonu veya eksojen genlerin entegrasyonu ile düzenleyen bir genetik mühendisliği tekniğidir. Prokaryotlardaki adaptif bağışıklıktan sorumlu olan CRISPR-Cas sisteminin keşfi ve bir genom düzenleme aracına dönüştürülmesi genetik mühendisliği alanında devrim etkisi yapmıştır. CRISPR-Cas sisteminde CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) “kümelenmiş düzenli aralıklı kısa palindromik tekrarlar” olarak adlandırılan bir seri DNA dizisini, Cas (CRISPR-associated protein) ise spesifik DNA zincirlerini tanımak ve kesmek için CRISPR dizilerini bir kılavuz gibi kullanan endonükleazları tanımlamaktadır. CRISPR-Cas teknolojisini, önceki tekniklerden farklı kılan, hemen her organizmanın genomuna kolaylıkla uygulanabilen hassas, verimli ve düşük maliyetli bir yöntem olmasıdır. Keşfinden günümüze kadar geçen süreçte bu teknolojinin tıp, biyomedikal, tarım ve hayvancılık gibi pek çok alanda kullanılabilecek umut verici bir araç olduğu kanıtlanmıştır. Öte yandan CRISPR-Cas teknolojisinin geniş uygulama potansiyeli, kolaylığı ve düşük maliyeti, kötü amaçlarla veya sorumsuzca kullanılma olasılığını artırmaktadır. Bu teknolojinin negatif yönlü kullanım olasılığı ve yaşanabilecek teknik başarısızlıklar, başta germ hattı genom düzenlemeleri olmak üzere birçok alandaki uygulamalarına yönelik etik ve ahlaki kaygıları artırmış ve biyogüvenlik tartışmalarını gündeme getirmiştir. CRISPR-Cas ve diğer genom düzenleme tekniklerinin kullanımına yönelik politikalar ülkeden ülkeye farklılık göstermekle birlikte birçok ülkede genom düzenlemelerini özel olarak ele alan yasal bir mevzuat henüz bulunmamakta veya geliştirilme aşamasındadır. Bu derleme çalışmasında, CRISPR-Cas teknolojisinin temel mekanizması açıklanarak tıp, biyomedikal, tarım ve hayvancılık gibi çeşitli alanlardaki uygulamalarına örnekler verilmiş ve potansiyel riskler ile farklı ülkelerdeki yasal düzenlemeler üzerinde durulmuştur.

Anahtar Kelimeler

• CRISPR-Cas
• etik
• potansiyel risk
• yasal düzenleme

CRISPR-CAS APPLICATIONS, POTENTIAL RISKS AND LEGAL ARRANGEMENTS

Öz

CRISPR-Cas technology is a genetic engineering technique that edits the genome of a living organisms by modification of endogenous genes or integration of exogenous genes. The discovery of the CRISPR-Cas system, which is responsible for adaptive immunity in prokaryotes, and its conversion into a genome editing tool revolutionized genetic engineering area. In the CRISPR-Cas system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defines a series of DNA sequences called "clustered regularly spaced short palindromic repeats" while Cas (CRISPR-associated protein) describes endonucleases that use CRISPR sequences as a guide to recognize and cleave specific strands of DNA. The difference between CRISPR-Cas technology from previous techniques is that it is a sensitive, efficient, and low-cost method that can be easily applied to the genome of almost all organisms. It has proven to be a promising tool that can be used in many areas such as medicine, biomedicine, agriculture, and animal husbandry, from its discovery to the present time. The wide application potential, simplicity, and low cost of CRISPR-Cas technology increase the possibility of malicious or irresponsible use. The possibility of negative use of this technology and potential technical failures has increased ethical and moral concerns about its applications in many areas, especially in germline genome editing, and made biosecurity discussions a current issue. Policies for the use of CRISPR-Cas and other genome editing techniques vary from country to country, but many countries do not yet have legislation that specifically for genome editing or is under development. In this review, the mechanism of the CRISPR-Cas system is described, examples of applications in medicine, biomedicine, agriculture, and animal husbandry are presented, and their potential risks and legal regulations in various countries are emphasized.

Anahtar Kelimeler

• CRISPR-Cas
• ethic
• potential risk
• regulation

Kaynakça

1. Ahmad, S., Shahzad, R., Jamil, S., Tabas-sum, J., Chaudhary, M. A. M., Atif, R. M., ... & Tang, S. (2021). Regulatory aspects, risk assessment, and toxicity associated with RNAi and CRISPR methods. In CRISPR and RNAi Systems (pp. 687-721). Elsevier.
2. Anderson, J. E., Michno, J. M., Kono, T. J., Stec, A. O., Campbell, B. W., Curtin, S. J., & Stupar, R. M. (2016). Genomic variation and DNA repair associated with soybean transgenesis: a comparison to cultivars and mutagenized plants. BMC biotechnology, 16(1), 1-13.
3. Anderson, K. R., Haeussler, M., Watana-be, C., Janakiraman, V., Lund, J., Modru-san, Z., ... & Warming, S. (2018). CRISPR off-target analysis in genetically engineered rats and mice. Nature methods, 15(7), 512-514.
4. Andersson, M., Turesson, H., Nicolia, A., Fält, A. S., Samuelsson, M., & Hofvander, P. (2017). Efficient targeted multiallelic mutagenesis in tetraploid potato (Sola-num tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant cell reports, 36(1), 117-128.
5. Aryal, N. K., Wasylishen, A. R., & Lozano, G. (2018). CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo. Cell death & disease, 9(11), 1-3.
6. Baltimore, D., Berg, P., Botchan, M., Carroll, D., Charo, R. A., Church, G., ... & Yamamoto, K. R. (2015). A prudent path forward for genomic engineering and germline gene modification. Science, 348(6230), 36-38.
7. Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature biotechnology, 34(9), 933-941.
8. Barrangou, R., & Marraffini, L. A. (2014). CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Molecular Cell, 54(2), 234-244.

9. Barrangou, R., & Notebaart, R. A. (2019). CRISPR-directed microbiome manipulation across the food supply chain. Trends in microbiology, 27(6), 489-496.
10. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., ... & Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315(5819), 1709-1712.
11. Bartkowski, B., Theesfeld, I., Pirscher, F., & Timaeus, J. (2018). Snipping around for food: economic, ethical and policy implications of CRISPR/Cas genome editing. Geoforum, 96, 172-180.
12. Baylis, F., Darnovsky, M., Hasson, K., & Krahn, T. M. (2020). Human germline and heritable genome editing: the global policy landscape. The CRISPR Journal, 3(5), 365-377.
13. Bikard, D., & Marraffini, L. A. (2012). Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Current Opinion in Immunology, 24(1), 15-20.
14. Bikard, D., Euler, C. W., Jiang, W., Nus-senzweig, P. M., Goldberg, G. W., Dupor-tet, X., ... & Marraffini, L. A. (2014). Exploiting CRISPR-Cas nucleases to pro-duce sequence-specific antimicrobials. Nature biotechnology, 32(11), 1146-1150.
15. Bogdanove, A. J., & Voytas, D. F. (2011). TAL effectors: customizable proteins for DNA targeting. Science333: 1843–1846. vivo effects of binding site variants.
16. Briner, A. E., & Barrangou, R. (2014). Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Applied and environmental microbiology, 80(3), 994-1001.
17. Briner, A. E., Lugli, G. A., Milani, C., Duranti, S., Turroni, F., Gueimonde, M., ... & Barrangou, R. (2015). Occurrence and diversity of CRISPR-Cas systems in the genus Bifidobacterium. PloS one, 10(7), e0133661.
18. Brokowski, C., & Adli, M. (2019). CRISPR ethics: moral considerations for applications of a powerful tool. Journal of molecular biology, 431(1), 88-101.
19. Brooks, C., Nekrasov, V., Lippman, Z. B., & Van Eck, J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant physiology, 166(3), 1292-1297.
20. Butiuc-Keul, A., Farkas, A., Carpa, R., & Iordache, D. (2021). CRISPR-Cas System: The Powerful Modulator of Accessory Genomes in Prokaryotes. Microbial Physiology, 1-16.
21. Cai, Y., Chen, L., Liu, X., Guo, C., Sun, S., Wu, C., ... & Hou, W. (2018). CRISPR/Cas9‐mediated targeted mutage-nesis of GmFT2a delays flowering time in soya bean. Plant biotechnology journal, 16(1), 176-185.
22. Carlson, D. F., Lancto, C. A., Zang, B., Kim, E. S., Walton, M., Oldeschulte, D., ... & Fahrenkrug, S. C. (2016). Production of hornless dairy cattle from genome-edited cell lines. Nature biotechnology, 34(5), 479-481.
23. Cermák, T., Baltes, N. J., Čegan, R., Zhang, Y., & Voytas, D. F. (2015). High-frequency, precise modification of the tomato genome. Genome biology, 16(1), 1-15.
24. Charo, R. A., & Greely, H. T. (2015). CRISPR critters and CRISPR cracks. The American Journal of Bioethics, 15(12), 11-17.
25. Citorik, R. J., Mimee, M., & Lu, T. K. (2014). Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nature biotechnology, 32(11), 1141-1145.
26. Coller, B. S. (2019). Ethics of human ge-nome editing. Annual Review of Medicine, 70, 289-305.
27. Crispo, M., Mulet, A. P., Tesson, L., Barrera, N., Cuadro, F., dos Santos-Neto, P. C., ... & Menchaca, A. (2015). Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PloS one, 10(8), e0136690.
28. Cyranoski, D. (2016). CRISPR gene-editing tested in a person for the first time. Nature news, 539(7630), 479.
29. Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., ... & Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607.
30. Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016). Beyond editing: repurposing CRISPR–Cas9 for precision genome regu-lation and interrogation. Nature reviews Molecular cell biology, 17(1), 5-15.
31. Dong, O. X., Yu, S., Jain, R., Zhang, N., Duong, P. Q., Butler, C., ... & Ronald, P. C. (2020). Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. Nature communications, 11(1), 1-10.
32. Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213).
33. Epstein, L. R., Lee, S. S., Miller, M. F., & Lombardi, H. A. (2021). CRISPR, ani-mals, and FDA oversight: Building a path to success. Proceedings of the National Academy of Sciences, 118(22), e2004831117.
34. Eriksson, D., Kershen, D., Nepomuceno, A., Pogson, B. J., Prieto, H., Purnhagen, K., ... & Whelan, A. (2019). A compari-son of the EU regulatory approach to di-rected mutagenesis with that of other jurisdictions, consequences for international trade and potential steps forward. New Phytologist, 222(4), 1673-1684.
35. Eş, I., Gavahian, M., Marti-Quijal, F. J., Lorenzo, J. M., Khaneghah, A. M., Tsat-sanis, C., ... & Barba, F. J. (2019). The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated challenges. Biotechnology advances, 37(3), 410-421.
36. Etaware, P. M. (2021). The effects of the phytochemistry of cocoa on the food chemistry of chocolate (s) and how disease resistance in cocoa can be improved using CRISPR/Cas9 technology. Food Chemistry: Molecular Sciences, 3, 100043.
37. Fan, Z., Mu, Y., Li, K., & Hackett, P. B. (2021). Safety evaluation of transgenic and genome-edited food animals. Trends in biotechnology. 40(4), 371-373.
38. Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D. L., Wei, P., ... & Zhu, J. K. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 23(10), 1229-1232.
39. Fineran, P. C., & Charpentier, E. (2012). Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology, 434(2), 202-209.
40. Fister, A. S., Landherr, L., Maximova, S. N., & Guiltinan, M. J. (2018). Transient expression of CRISPR/Cas9 machinery targeting TcNPR3 enhances defense response in Theobroma cacao. Frontiers in plant science, 9, 268.
41. FDA (2017). US Food and Drug Admi-nistration. Regulation of Intentionally Altered Genomic DNA in Animals, Draft Guidance for Industry. Rockville: US Food and Drug Administration.
42. FDA (2022). (Food and Drug Administra-tion). Risk Assessment Summary – V-006378 PRLR-SLICK cattle. https://www.fda.gov/media/155706/download. Erişim tarihi: 3 Ekim 2022.
43. Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology, 31(9), 822-826.
44. Gao, Y., Wu, H., Wang, Y., Liu, X., Chen, L., Li, Q., ... & Zhang, Y. (2017). Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects. Genome biology, 18(1), 1-15.
45. Garneau, J. E., Dupuis, M. È., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., ... & Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67-71.
46. Globus, R., & Qimron, U. (2018). A technological and regulatory outlook on CRISPR crop editing. Journal of cellular biochemistry, 119(2), 1291-1298.
47. Gomaa, A. A., Klumpe, H. E., Luo, M. L., Selle, K., Barrangou, R., & Beisel, C. L. (2014). Programmable removal of bacte-rial strains by use of genome-targeting CRISPR-Cas systems. MBio, 5(1), e00928-13.
48. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B., & Taipale, J. (2018). CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature medicine, 24(7), 927-930.
49. He, Z., Zhang, T., Jiang, L., Zhou, M., Wu, D., Mei, J., & Cheng, Y. (2018). Use of CRISPR/Cas9 technology efficiently targetted goat myostatin through zygotes microinjection resulting in double-muscled phenotype in goats. Bioscience reports, 38(6). BSR20180742.
50. Hellmich, R., Sid, H., Lengyel, K., Flisi-kowski, K., Schlickenrieder, A., Bartsch, D., ... & Schusser, B. (2020). Acquiring resistance against a retroviral infection via CRISPR/Cas9 targeted genome editing in a commercial chicken li-ne. Frontiers in genome editing, 2, 3.
51. Hermans, P. W., Van Soolingen, D., Bik, E. M., De Haas, P. E., Dale, J. W., & Van Embden, J. D. (1991). Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infection and Immunity, 59(8), 2695-2705.
52. Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., ... & Kohn, D. B. (2016). CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells. Molecular Therapy, 24(9), 1561-1569.
53. Horvath, P., Romero, D. A., Coûté-Monvoisin, A. C., Richards, M., Deveau, H., Moineau, S., ... & Barrangou, R. (2008). Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. Journal of bacteriology, 190(4), 1401-1412.
54. Hullahalli, K., Rodrigues, M., Schmidt, B. D., Li, X., Bhardwaj, P., & Palmer, K. L. (2015). Comparative analysis of the orphan CRISPR2 locus in 242 Enterococcus faecalis strains. PloS one, 10(9), e0138890.
55. Ishii, T. (2017). Genome-edited livestock: Ethics and social acceptance. Animal Frontiers, 7(2), 24-32.
56. Ishii, T., & Araki, M. (2016). Consumer acceptance of food crops developed by genome editing. Plant cell reports, 35(7), 1507-1518.
57. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of bacteriology, 169(12), 5429-5433.
58. Jansen, R., Embden, J. D. V., Gaastra, W., & Schouls, L. M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology, 43(6), 1565-1575.
59. Jiang, F., & Doudna, J. A. (2015). The structural biology of CRISPR-Cas systems. Current opinion in structural biology, 30, 100-111.
60. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821.
61. Kalidasan, V., & Theva Das, K. (2021). Is Malaysia Ready for Human Gene Editing: A Regulatory, Biosafety and Biosecurity Perspective. Frontiers in Bioengineering and Biotechnology, 9, 649203.
62. Kuluev, B. R., Gumerova, G. R., Mikhay-lova, E. V., Gerashchenkov, G. A., Rozhnova, N. A., Vershinina, Z. R., ... & Chemeris, A. V. (2019). Delivery of CRISPR/Cas components into higher plant cells for genome editing. Russian Journal of Plant Physiology, 66(5), 694-706.
63. Kurtz, S., & Petersen, B. (2019). Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing. Theriogenology, 137, 67-74.
64. Lander, E. S., Baylis, F., Zhang, F., Charpentier, E., Berg, P., Bourgain, C., ... & Winnacker, E. L. (2019). Adopt a moratorium on heritable genome editing. Nature,567, 165-168.
65. Lassoued, R., Macall, D. M., Smyth, S. J., Phillips, P. W., & Hesseln, H. (2019). Risk and safety considerations of genome edited crops: expert opinion. Current Research in Biotechnology, 1, 11-21.
66. Ledford, H. (2015). CRISPR, the disruptor. Nature, 522(7544), 20-25.
67. Leung, R. K. K., Cheng, Q. X., Wu, Z. L., Khan, G., Liu, Y., Xia, H. Y., & Wang, J. (2021). CRISPR-Cas12-based nucleic acids detection systems. Methods, 203, 276,281
68. Li, G., Liu, Y.G. and Chen, Y. (2019c) Genome-editing technologies: the gap between application and policy. Science China Life Sciences, 62, 1534-1538.
69. Li, J. , Manghwar, H. , Sun, L., Wang, P. , Wang, G., Sheng, H., …... & Zhang, X. (2019b) Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants. Plant Biotechnology Journal, 17 (5), 858–868.
70. Li, J. F., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., ... & Sheen, J. (2013). Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology, 31(8), 688-691.
71. Li, Y., Li, S., Wang, J., & Liu, G. (2019a). CRISPR/Cas systems towards next-generation biosensing. Trends in biotechnology, 37(7), 730-743.
72. Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., ... & Huang, J. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & cell, 6(5), 363-372.
73. Liang, Z., Zhang, K., Chen, K., & Gao, C. (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of Genetics and Genomics, 41(2), 63-68.
74. Liu, X., Wang, Y., Tian, Y., Yu, Y., Gao, M., Hu, G., ... & Zhang, Y. (2014). Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases. Proceedings of the Royal Society B: Biological Sciences, 281(1780), 20133368.
75. Ma, H., Marti-Gutierrez, N., Park, S. W., Wu, J., Lee, Y., Suzuki, K., ... & Mitali-pov, S. (2017). Correction of a pathogenic gene mutation in human embryos. Nature, 548(7668), 413-419.
76. Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E., ... & Mills, D. (2006). Comparative genomics of the lactic acid bacteria. Proceedings of the National Academy of Sciences, 103(42), 15611-15616.
77. Manghwar, H., Li, B., Ding, X., Hussain, A., Lindsey, K., Zhang, X., & Jin, S. (2020). CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off‐target evaluation, and strategies to mitigate off‐target effects. Advanced science, 7(6), 1902312.
78. Marangi, M., & Pistritto, G. (2018). Innovative therapeutic strategies for cystic fibrosis: moving forward to CRISPR technique. Frontiers in pharmacology, 9, 396.
79. Mehravar, M., Shirazi, A., Nazari, M., & Banan, M. (2019). Mosaicism in CRISPR/Cas9-mediated genome editing. Developmental biology, 445(2), 156-162.
80. Memi, F., Ntokou, A., & Papangeli, I. (2018, December). CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations. In Seminars in perinatology (Vol. 42, No. 8, pp. 487-500). WB Saunders.
81. Mojica, F. J., Díez‐Villaseñor, C., Soria, E., & Juez, G. (2000). Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular Microbiology, 36(1), 244-246.
82. Mojica, F. J., García-Martínez, J., & So-ria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of molecular evolution, 60(2), 174-182.
83. Mojica, F. J., Juez, G., & Rodriguez‐Valera, F. (1993). Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Molecular Microbiology, 9(3), 613-621.
84. Mubarik, M. S., Khan, S. H., & Sajjad, M. (2021). Key Applications of CRISPR/Cas for Yield and Nutritional Improvement. In CRISPR Crops (pp. 213-230). Springer, Singapore.
85. Nakata, A. T. S. U. O., Amemura, M. I. T. S. U. K. O., & Makino, K. O. Z. O. (1989). Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome. Journal of bacteriology, 171(6), 3553-3556.
86. NASEM (2017). National Academies of Sciences, Engineering and Medicine. Human Genome Editing: Science, Ethics, and Governance. Washington, DC: The National Academies Press.
87. Ni, W., Qiao, J., Hu, S., Zhao, X., Regouski, M., Yang, M., Chen, C., 2014. Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS ONE, 9(9), 1–8.
88. Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., ... & Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 156(5), 935-949.
89. Normile, D. (2018). Shock greets claim of CRISPR-edited babies. Science, 362(6418), 978-979.
90. Oishi, I., Yoshii, K., Miyahara, D., & Ta-gami, T. (2018). Efficient production of human interferon beta in the white of eggs from ovalbumin gene–targeted hens. Scientific reports, 8(1), 1-12.
91. Ormond, K. E., Mortlock, D. P., Scholes, D. T., Bombard, Y., Brody, L. C., Faucett, W. A., ... & Young, C. E. (2017). Human germline genome editing. The American Journal of Human Genetics, 101(2), 167-176.
92. Ouyang, B., Gu, X., & Holford, P. (2017). Plant genetic engineering and biotechnology: a sustainable solution for future food security and industry. Plant Growth Regulation, 83(2), 171-173.
93. Peng, J., Wang, Y., Jiang, J., Zhou, X., Song, L., Wang, L., ... & Zhang, P. (2015). Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Scientific reports, 5(1), 1-6.
94. Petersen B, Niemann H (2015). Molecular scissors and their application in genetically modified farm animals. Transgenic research, 24(3), 381-396.
95. Pfeiffer, M., Quétier, F., & Ricroch, A. (2018). Genome editing in agricultural biotechnology. In Advances in Botanical Research (Vol. 86, pp. 245-286). Academic Press.
96. Pickar-Oliver, A., & Gersbach, C. A. (2019). The next generation of CRISPR–Cas technologies and applications. Nature reviews Molecular cell biology, 20(8), 490-507.
97. Pourcel, C., Salvignol, G., & Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary stu-dies. Microbiology, 151(3), 653-663.
98. Proudfoot, C., Lillico, S., & Tait-Burkard, C. (2019). Genome editing for disease resistance in pigs and chickens. Animal Frontiers, 9(3), 6-12.
99. Pyott, D. E., Sheehan, E., & Molnar, A. (2016). Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants. Molecular plant pathology, 17(8), 1276-1288.
100. Ran, F. A., Hsu, P. D., Lin, C. Y., Goo-tenberg, J. S., Konermann, S., Trevino, A. E., ... & Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154(6), 1380-1389.
101. Rath, D., Amlinger, L., Rath, A., & Lundgren, M. (2015). The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie, 117, 119-128.
102. Rautela, I., Uniyal, P., Thapliyal, P., Cha-uhan, N., Sinha, V. B., & Sharma, M. D. (2021). An extensive review to facilitate understanding of CRISPR technology as a gene editing possibility for enhanced therapeutic applications. Gene, 145615.
103. Raza, S. H. A., Hassanin, A. A., Pant, S. D., Bing, S., Sitohy, M. Z., Abdelnour, S. A., ... & Zan, L. (2021). Potentials, pros-pects and applications of genome editing technologies in livestock production. Saudi Journal of Biological Sciences, 29(4), 1928-1935
104. Regalado, A. (2015). Engineering the perfect baby. MITS Technol Rev, 118(3), 27-33.
105. Regalado, A. (2016). Top US intelligence official calls gene editing a WMD threat. MIT Technology Review.
106. Ren, C., Liu, X., Zhang, Z., Wang, Y., Duan, W., Li, S., & Liang, Z. (2016). CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific reports, 6(1), 1-9.
107. Ricroch, A., Clairand, P., & Harwood, W. (2017). Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture. Emerging Topics in Life Sciences, 1(2), 169-182.
108. Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E., & Lippman, Z. B. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell, 171(2), 470-480.
109. Ruan, J., Xu, J., Chen-Tsai, R. Y., & Li, K. (2017). Genome editing in livestock: Are we ready for a revolution in animal breeding industry?. Transgenic research, 26(6), 715-726.
110. Sanozky-Dawes, R., Selle, K., O'Flaherty, S., Klaenhammer, T., & Barrangou, R. (2015). Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri. Microbiology, 161(9), 1752-1761.
111. Sarchet, P., & Le Page, M. (2015). Star-ting gun fired on gene editing. Newscientists, 226(3019), 8-9.
112. Schulman, A. H., Oksman‐Caldentey, K. M., & Teeri, T. H. (2020). European Court of Justice delivers no justice to Europe on genome‐edited crops. Plant biotechnology journal, 18(1), 8.
113. Selle, K., & Barrangou, R. (2015). CRISPR‐Based technologies and the future of food science. Journal of food science, 80(11), R2367-R2372.
114. Shi, J., Gao, H., Wang, H., Lafitte, H. R., Archibald, R. L., Yang, M., ... & Habben, J. E. (2017). ARGOS 8 variants generated by CRISPR‐Cas9 improve maize grain yield under field drought stress conditions. Plant biotechnology journal, 15(2), 207-216.
115. Siegrist, M. (2000). The influence of trust and perceptions of risks and benefits on the acceptance of gene technology. Risk analysis, 20(2), 195-204.
116. Smith, K. R. (2003). Gene therapy: theoretical and bioethical concepts. Archives of medical research, 34(4), 247-268.
117. Sorek, R., Kunin, V., & Hugenholtz, P. (2008). CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology, 6(3), 181-186.
118. Stephens, C. J., Lauron, E. J., Kashentse-va, E., Lu, Z. H., Yokoyama, W. M., & Curiel, D. T. (2019). Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. Journal of Controlled Release, 298, 128-141.
119. Stout, E., Klaenhammer, T., & Barran-gou, R. (2017). CRISPR-Cas technologies and applications in food bacteria. Annual review of food science and technology, 8, 413-437.
120. Sun, Y., Jiao, G., Liu, Z., Zhang, X., Li, J., Guo, X., ... & Xia, L. (2017). Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in plant science, 8, 298.
121. Sun, Y., Zhang, X., Wu, C., He, Y., Ma, Y., Hou, H., ... & Xia, L. (2016). Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate syntha-se. Molecular plant, 9(4), 628-631.
122. Tang, X. D., Gao, F., Liu, M. J., Fan, Q. L., Chen, D. K., & Ma, W. T. (2019). Methods for enhancing clustered regu-larly interspaced short palindromic repeats/Cas9-mediated homology-directed repair efficiency. Frontiers in Genetics, 10, 551.
123. Turnbull, C., Lillemo, M., & Hvoslef-Eide, T. A. (2021). Global regulation of genetically modified crops amid the gene edited crop boom–a review. Frontiers in Plant Science, 12, 630396.
124. Valletta, S., Dolatshad, H., Bartenstein, M., Yip, B. H., Bello, E., Gordon, S., ... & Boultwood, J. (2015). ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenog-rafts. Oncotarget, 6(42), 44061.
125. Van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., & Brouns, S. J. (2009). CRISPR-based adaptive and heritable immunity in prokaryotes. Trends in biochemical sciences, 34(8), 401-407.
126. Van Eenennaam, A. L. (2018). The importance of a novel product risk-based trigger for gene-editing regulation in food animal species. The CRISPR Journal, 1(2), 101-106.
127. Van Eenennaam, A. L. (2019, August). Application of genome editing in farm animals: Cattle. In Transgenic Research (Vol. 28, No. 2, pp. 93-100). Sprin-ger International Publishing.
128. Van Eenennaam, A. L., & Mueller, M. L. (2022). CURRENT STATE OF GENOME EDITING AND WHAT IT MEANS TO BEEF PRODUCERS. Proceedings, Applied Reproductive Strategies in Beef Cattle August 30-31, 2022; San Antonio, TX
129. Waltz, E. (2016). Gene-edited CRISPR mushroom escapes US regulation. Nature News, 532(7599), 293.
130. Waltz, E. (2018). With a free pass, CRISPR-edited plants reach market in record time. Nature biotechnology, 36(1), 6-8.
131. Wang, T., Zhang, H., & Zhu, H. (2019). CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Horticulture research, 6.
132. Wang, W., Pan, Q., He, F., Akhunova, A., Chao, S., Trick, H., & Akhunov, E. (2018). Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. The CRISPR journal, 1(1), 65-74.
133. Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J. L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature biotechnology, 32(9), 947-951.
134. Wang, Y., Geng, L., Yuan, M., Wei, J., Jin, C., Li, M., ... & Li, X. (2017). Deletion of a target gene in Indica rice via CRISPR/Cas9. Plant Cell Reports, 36(8), 1333-1343.
135. Wani, A. K., Akhtar, N., & Shukla, S. (2022). CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use. Forensic science internatio-nal, 111274.
136. Weber, J., Öllinger, R., Friedrich, M., Ehmer, U., Barenboim, M., Steiger, K., ... & Rad, R. (2015). CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proceedings of the National Aca-demy of Sciences, 112(45), 13982-13987.
137. Wei, J., Wagner, S., Lu, D., Maclean, P., Carlson, D. F., Fahrenkrug, S. C., & Laib-le, G. (2015). Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome. Scientific reports, 5(1), 1-12.
138. Whitworth, K. M., Rowland, R. R., Ewen, C. L., Trible, B. R., Kerrigan, M. A., Cino-Ozuna, A. G., ... & Prather, R. S. (2016). Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature biotechno-logy, 34(1), 20-22.
139. Whitworth, K. M., Rowland, R. R., Petro-van, V., Sheahan, M., Cino-Ozuna, A. G., Fang, Y., ... & Prather, R. S. (2019). Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic research, 28(1), 21-32.
140. Wilkinson, R., & Wiedenheft, B. (2014). A CRISPR method for genome engineering. F1000prime reports, 6.
141. Wray-Cahen, D., Bodnar, A., Rexroad, C., Siewerdt, F., & Kovich, D. (2022). Ad-vancing genome editing to improve the sustainability and resiliency of animal agriculture. CABI Agriculture and Bioscience, 3(1), 1-17.
142. Wright, A. V., Nuñez, J. K., & Doudna, J. A. (2016). Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell, 164(1-2), 29-44.
143. Xie, S., Ji, Z., Suo, T., Li, B., & Zhang, X. (2021). Advancing sensing technology with CRISPR: From the detection of nuc-leic acids to a broad range of analytes–A review. Analytica Chimica Acta, 1185, 338848.
144. Xu, Z. S., Yang, Q. Q., Feng, K., & Xiong, A. S. (2019). Changing carrot color: insertions in DcMYB7 alter the regu-lation of anthocyanin biosynthesis and modification. Plant physiology, 181(1), 195-207.
145. Yeh, Y. C., Kinoshita, M., Ng, T. H., Chang, Y. H., Maekawa, S., Chiang, Y. A., ... & Wang, H. C. (2017). Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes). Scientific reports, 7(1), 1-13.
146. Yunes, M. C., Teixeira, D. L., von Keyserlingk, M. A., & Hötzel, M. J. (2019). Is gene editing an acceptable alternative to castration in pigs?. PloS one, 14(6), e0218176.
147. Zhang, B. (2021). CRISPR/Cas gene therapy. Journal of Cellular Physiology, 236(4), 2459-2481.
148. Zhang, D., Hussain, A., Manghwar, H., Xie, K., Xie, S., Zhao, S., ... & Ding, F. (2020). Genome editing with the CRISPR‐Cas system: an art, ethics and global regulatory perspective. Plant biotechnology journal, 18(8), 1651-1669.
149. Zhang, Y., Wu, Y., Wu, Y., Chang, Y., & Liu, M. (2021). CRISPR-Cas systems: from gene scissors to programmable biosensors. TrAC Trends in Analytical Che-mistry, 137, 116210.
150. Zhou, J., Peng, Z., Long, J., Sosso, D., Liu, B. O., Eom, J. S., ... & Yang, B. (2015). Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. The Plant Journal, 82(4), 632-643.
151. Zhou, J., Xin, X., He, Y., Chen, H., Li, Q., Tang, X., ... & Zhang, Y. (2019). Multiplex QTL editing of grain-related genes improves yield in elite rice varieties. Plant cell reports, 38(4), 475-485.
152. Zischewski, J., Fischer, R., & Bortesi, L. (2017). Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnology advances, 35(1), 95-104.