Aplicaciones clínicas de la herramienta CRISPR-Cas

Autores/as

DOI:

https://doi.org/10.51481/amc.v65i3.1268

Palabras clave:

CRISPR-Cas, edición genética, terapia génica, ZFN, aplicaciones clínicas

Resumen

El desarrollo de tecnologías para la edición del genoma ha abierto la posibilidad de apuntar directamente y modificar secuencias genómicas en casi todo tipo de células eucariotas.
La edición del genoma ha ampliado nuestra capacidad para dilucidar la contribución de la genética a las enfermedades al promover la creación de modelos celulares y animales más precisos de procesos patológicos y ha comenzado a mostrar su potencial en una variedad de campos, que van desde la investigación básica hasta la biotecnología aplicada y biomédica. Entre estas tecnologías, el uso de las repeticiones palindrómicas cortas agrupadas regularmente espaciadas ha acelerado, en gran medida, el progreso de la edición de genes desde el concepto hasta la práctica clínica, generando, además, interés debido, no solo a su precisión y eficiencia, sino también a la rapidez y a los costos necesarios para su implementación en comparación con otras tecnologías de edición genómica.

En esta revisión se presenta información recabada de publicaciones indexadas en la base de datos PubMed que se encontraron mediante el uso de palabras claves asociadas con la tecnología y que se filtraron para retener solo aquellas con evidencias de avances clínicamente relevantes y que permiten demostrar algunas de las aplicaciones que tiene esta tecnología en la investigación, pronóstico y tratamiento de enfermedades genéticas, cardiovasculares, virales, entre otras; esto con el objetivo de dar a conocer la situación actual de los avances en aplicaciones clínicas de la herramienta CRISPR-Cas y fomentar aún más la investigación en esta tecnología, la cual, tal como se evidencia a lo largo de esta revisión, posee una gran versatilidad y un amplio rango de aplicaciones, lo que ofrece una enorme oportunidad en el campo de la medicina genómica, pero que, a su vez, requiere un mayor fomento en su investigación para mejorar la tecnología y acercarla aún más a consolidar aplicaciones clínicas de uso seguro, confiable y consistente.

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Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science. 1996; 273:1516–7. DOI: 10.1126/science.273.5281.1516

Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE, et al. A polymor-phic DNA marker genetically linked to Huntington’s disease. Nature. 1983; 306:234–8. DOI 10.1038/306234a0

Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015; 21:121–31. DOI: 10.1038/nm.3793

Roth SC. What is genomic medicine? J Med Libr Assoc. 2019; 107:3. DOI: 10.5195/jmla.2019.604

Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Mol Ther. 2016; 24:430–46.DOI: 10.1038/mt.2016.10

Kaufmann KB, Büning H, Galy A, Schambach A, Grez M. Gene therapy on the move. EMBO Mol Med. 2013; 5:1642–61. DOI: 10.1002/emmm.201202287

Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest. 2014; 124:4154–61. DOI: 10.1172/jci72992

Lee SH, Kim S, Hur JK. CRISPR and target-specific DNA endonucleases for efficient DNA knock-in in eukaryotic genomes. Mol Cells. 2018; 41:943–52. DOI: 10.14348/molcells.2018.0408

O’Driscoll M, Jeggo PA. The role of double-strand break repair - insights from human genetics. Nat Rev Genet. 2006; 7:45–54. DOI: 10.1038/nrg1746

Rothstein RJ. [12] One-step gene disruption in yeast. En: Recombinant DNA Part C. Else-vier; 1983. p. 202–11.

Diakun GP, Fairall L, Klug A. EXAFS study of the zinc-binding sites in the protein tran-scription factor IIIA. Nature. 1986; 324:698–9. DOI: 10.1038/324698a0

Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim Y-G, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol. 2001; 21:289–97. DOI: 10.1128/mcb.21.1.289-297.2001

Mojarrad M, Bozorg Qomi S, Asghari A. An overview of the crispr-based genomic- and epigenome-editing system: Function, applications, and challenges. Adv Biomed Res. 2019; 8:49. DOI: 10.4103/abr.abr_41_19

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009; 326:1509–12. DOI: 10.1126/science.1178811

Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010; 186:757–61. DOI: 10.1534/genetics.110.120717

Mussolino C, Morbitzer R, Lütge F, Dannemann N, Lahaye T, Cathomen T. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucle-ic Acids Res. 2011; 39:9283–93. DOI: 10.1093/nar/gkr597

Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C, et al. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 2013; 41:e63–e63. DOI: 10.1093/nar/gks1446

Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011; 29:731–4. DOI: 10.1038/nbt.1927

Ashmore-Harris C, Fruhwirth GO. The clinical potential of gene editing as a tool to engi-neer cell-based therapeutics. Clin Transl Med. 2020; 9:15. DOI: 10.1186/s40169-020-0268-z

González-Romero E, Martínez-Valiente C, García-Ruiz C, Vázquez-Manrique RP, Cervera J, Sanjuan-Pla A. CRISPR to fix bad blood: a new tool in basic and clinical hematology. Hae-matologica. 2019; 104:881–93. DOI: 10.3324/haematol.2018.211359

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. J Bacteriol. 1987; 169:5429–33. DOI: 10.1128/jb.169.12.5429-5433.1987

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–21. DOI: 10.1126/science.1225829

Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palin-drome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005; 151:2551–61. DOI:10.1099/mic.0.28048-0

Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new re-peats by preferential uptake of bacteriophage DNA, and provide additional tools for evolution-ary studies. Microbiology. 2005; 151:653–63. DOI: 10.1099/mic.0.27437-0

Barrangou R. The roles of CRISPR–Cas systems in adaptive immunity and beyond. Curr Opin Immunol. 2015; 32:36–41. DOI: 10.1016/j.coi.2014.12.008

Jacinto FV, Link W, Ferreira BI. CRISPR/Cas9‐mediated genome editing: From basic re-search to translational medicine. J Cell Mol Med. 2020; 24:3766–78. DOI: 10.1111/jcmm.14916

Cornu TI, Mussolino C, Cathomen T. Refining strategies to translate genome editing to the clinic. Nat Med. 2017; 23:415–23. DOI: 10.1038/nm.4313

Posey JE. Genome sequencing and implications for rare disorders. Orphanet J Rare Dis. 2019; 14:153. DOI: 10.1186/s13023-019-1127-0

Ghosh D, Venkataramani P, Nandi S, Bhattacharjee S. CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics. Cancer Cell Int. 2019; 19(1). DOI: 10.1186/s12935-019-0726-0

Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, et al. Saccharo-myces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 2012; 40:D700–5. DOI: 10.1093/nar/gkr1029

Karginov FV, Hannon GJ. The CRISPR system: Small RNA-guided defense in bacteria and Archaea. Mol Cell. 2010; 37:7–19. DOI: 10.1016/j.molcel.2009.12.033

Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al. An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol. 2015; 13:722–36. DOI: 10.1038/nrmicro3569

Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial ge-nomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:233–9. DOI: 10.1038/nbt.2508

Capecchi MR. Altering the genome by homologous recombination. Science. 1989; 244:1288–92. DOI: 10.1126/science.2660260

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013; 8:2281–308. DOI: 10.1038/nprot.2013.143

Gaj T, Gersbach CA, Barbas CF III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013; 31:397–405. DOI: 10.1016/j.tibtech.2013.04.004

Feng S, Hu L, Zhang Q, Zhang F, Du J, Liang G, et al. CRISPR/Cas technology promotes the various application of Dunaliella salina system. Appl Microbiol Biotechnol. 2020; 104:8621–30. DOI: 10.1007/s00253-020-10892-6

Zhan T, Rindtorff N, Betge J, Ebert MP, Boutros M. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol. 2019; 55:106–19. DOI: 10.1016/j.semcancer.2018.04.001

Zhang H, Qin C, An C, Zheng X, Wen S, Chen W, et al. Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer. Mol Cancer. 2021; 20:1. DOI: 10.1186/s12943-021-01431-6

Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, et al. Gen-eration of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol. 2014; 32:941–6. DOI: 10.1038/nbt.2951

Maji S, Panda S, Samal SK, Shriwas O, Rath R, Pellecchia M, et al. Bcl-2 Antiapoptotic Family Proteins and Chemoresistance in Cancer. En: Advances in Cancer Research. Elsevier; 2018. p. 37–75.

Gao W, Zhang Y, Luo H, Niu M, Zheng X, Hu W, et al. Targeting SKA3 suppresses the proliferation and chemoresistance of laryngeal squamous cell carcinoma via impairing PLK1–AKT axis-mediated glycolysis. Cell Death Dis. 2020; 11:10. DOI: 10.1038/s41419-020-03104-6

Yu J, Zhou J, Xu F, Bai W, Zhang W. High expression of Aurora-B is correlated with poor prognosis and drug resistance in non-small cell lung cancer. Int J Biol Markers. 2018; 33:215–21. DOI: 10.1177/1724600817753098

Liu Y-P, Ling Y, Qi Q-F, Zhang Y-P, Zhang C-S, Zhu C-T, et al. The effects of ERCC1 expression levels on the chemosensitivity of gastric cancer cells to platinum agents and survival in gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy. Oncol Lett. 2013; 5:935–42. DOI: 10.3892/ol.2012.1096

Li AH, Morrison AC, Kovar C, Cupples LA, Brody JA, Polfus LM, et al. Analysis of loss-of-function variants and 20 risk factor phenotypes in 8,554 individuals identifies loci influenc-ing chronic disease. Nat Genet. 2015; 47:640–2. DOI: 10.1038/ng.3270

Abrahimi P, Chang WG, Kluger MS, Qyang Y, Tellides G, Saltzman WM, et al. Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9. Circ Res. 2015; 117:121–8. DOI: 10.1161/CIRCRESAHA.117.306290

Schlossarek S, Mearini G, Carrier L. Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: Mechanisms and therapeutic opportunities. J Mol Cell Cardiol. 2011; 50:613–20. DOI: 10.1016/j.yjmcc.2011.01.014

Ma H, Marti-Gutierrez N, Park S-W, Wu J, Lee Y, Suzuki K, et al. Correction of a patho-genic gene mutation in human embryos. Nature. 2017; 548:413–9. DOI: 10.1038/nature23305

Sun J, Wang J, Zheng D, Hu X. Advances in therapeutic application of CRISPR-Cas9. Brief Funct Genomics. 2020; 19:164–74. DOI:10.1093/bfgp/elz031

Min Y-L, Bassel-Duby R, Olson EN. CRISPR correction of duchenne muscular dystrophy. Annu Rev Med. 2019; 70:239–55. DOI: 10.1146/annurev-med-081117-010451

Bjursell M, Porritt MJ, Ericson E, Taheri-Ghahfarokhi A, Clausen M, Magnusson L, et al. Therapeutic genome editing with CRISPR/Cas9 in a humanized mouse model ameliorates α1-antitrypsin deficiency phenotype. E Bio Medicine. 2018; 29:104–11. DOI: 10.1016/j.ebiom.2018.02.015

Wang D, Zhang G, Gu J, Shao X, Dai Y, Li J, et al. In vivo generated hematopoietic stem cells from genome edited induced pluripotent stem cells are functional in platelet-targeted gene therapy of murine hemophilia A. Haematologica. 2020; 105:e175–9. DOI: 10.3324/haematol.2019.219089

György B, Nist-Lund C, Pan B, Asai Y, Karavitaki KD, Kleinstiver BP, et al. Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss. Nat Med. 2019; 25:1123–30. DOI: 10.1038/s41591-019-0500-9

Khosravi MA, Abbasalipour M, Concordet J-P, Berg JV, Zeinali S, Arashkia A, et al. Tar-geted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease. Eur J Pharmacol. 2019; 854:398–405. DOI: 10.1016/j.ejphar.2019.04.042

Antonarakis SE, Kazazian HH Jr, Orkin SH. DNA polymorphism and molecular pathology of the human globin gene clusters. Hum Genet. 1985; 69:1–14. DOI: 10.1007/bf00295521

Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing tech-nology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020; 5. DOI: 10.1038/s41392-019-0089-y

Raaijmakers RHL, Ripken L, Ausems CRM, Wansink DG. CRISPR/Cas applications in myotonic dystrophy: Expanding opportunities. Int J Mol Sci. 2019; 20:3689. DOI: 10.3390/ijms20153689

Mahdieh N, Rabbani B. Beta thalassemia in 31,734 cases with HBB gene mutations: Path-ogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East. Blood Rev. 2016; 30:493–508. DOI: 10.1016/j.blre.2016.07.001

Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature. 2016; 539:384–9. DOI: 10.1038/nature20134

Hultquist JF, Schumann K, Woo JM, Manganaro L, McGregor MJ, Doudna J, et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV-host interactions in primary human T cells. Cell Rep. 2016; 17:1438–52. DOI: 10.1016/j.celrep.2016.09.080

Rivella S, Rachmilewitz E. Future alternative therapies for β-thalassemia. Expert Rev He-matol. 2009; 2:685–97. DOI: 10.1586/ehm.09.56

Isgrò A, Gaziev J, Sodani P, Lucarelli G. Progress in hematopoietic stem cell transplanta-tion as allogeneic cellular gene therapy in thalassemia. Ann N Y Acad Sci. 2010; 1202:149–54. DOI: 10.1111/j.1749-6632.2010.05543.x

Hanna J, Wernig M, Markoulaki S, Sun C-W, Meissner A, Cassady JP, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007; 318:1920–3. DOI: 10.1126/science.1152092

Park SH, Lee CM, Dever DP, Davis TH, Camarena J, Srifa W, et al. Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease. Nucleic Acids Res [Internet]. 2019; 47:7955–72. DOI: 10.1093/nar/gkz475

Song B, Fan Y, He W, Zhu D, Niu X, Wang D. Improved Hematopoietic Differentiation Efficiency of Gene-Corrected Beta-Thalassemia Induced Pluripotent Stem Cells by CRISPR/Cas9 System. Stem Cells Dev. 2015; 24:1053–65. DOI: 10.1089/scd.2014.0347

Lee JK, Jeong E, Lee J, Jung M, Shin E, Kim Y-H, et al. Directed evolution of CRISPR-Cas9 to increase its specificity. Nat Commun. 2018; 9. DOI: 10.1038/s41467-018-05477-x

Fan H-C, Chi C-S, Lee Y-J, Tsai J-D, Lin S-Z, Harn H-J. The role of gene editing in neuro-degenerative diseases. Cell Transplant. 2018; 27:364–78. DOI: 10.1177/0963689717753378

Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020; 38:870–4. DOI: 10.1038/s41587-020-0513-4

Mohammadzadeh I, Qujeq D, Yousefi T, Ferns GA, Maniati M, Vaghari-Tabari M. CRISPR/Cas9 gene editing: A new therapeutic approach in the treatment of infection and auto-immunity. IUBMB Life. 2020; 72:1603–21. DOI: 10.1002/iub.2296

Sension MG. Long-term suppression of HIV infection: Benefits and limitations of current treatment options. J Assoc Nurses AIDS Care. 2007; 18:S2–10. DOI: 10.1016/j.jana.2006.11.012

Ruelas DS, Greene WC. An integrated overview of HIV-1 latency. Cell. 2013; 155:519–29. DOI: 10.1016/j.cell.2013.09.044

Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med. 2003; 9:727–8. DOI: 10.1038/nm880

Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep. 2013; 3. DOI: 10.1038/srep02510

Yin C, Zhang T, Qu X, Zhang Y, Putatunda R, Xiao X, et al. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Mol Ther. 2017;25:1168–86. DOI: 10.1016/j.ymthe.2017.03.012

Liao H-K, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, et al. Use of the CRISPR/Cas9 sys-tem as an intracellular defense against HIV-1 infection in human cells. Nat Commun. 2015; 6:6413. DOI: 10.1038/ncomms7413

Venkatakrishnan B, Zlotnick A. The structural biology of hepatitis B virus: Form and func-tion. Annu Rev Virol. 2016; 3:429–51. DOI: 10.1146/annurev-virology-110615-042238

Yuen M-F, Chen D-S, Dusheiko GM, Janssen HLA, Lau DTY, Locarnini SA, et al. Hepati-tis B virus infection. Nat Rev Dis Primers. 2018; 4:18035. DOI: 10.1038/nrdp.2018.35

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015; 6:363–72. DOI: 10.1007/s13238-015-0153-

Moyo B, Bloom K, Scott T, Ely A, Arbuthnot P. Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus. Virus Res. 2018; 244:311–20. DOI: 10.1016/j.virusres.2017.01.003

Kennedy EM, Bassit LC, Mueller H, Kornepati AVR, Bogerd HP, Nie T, et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology. 2015; 476:196–205. DOI: 10.1016/j.virol.2014.12.001

Ramanan V, Shlomai A, Cox DBT, Schwartz RE, Michailidis E, Bhatta A, et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep. 2015; 5. DOI: 10.1038/srep10833

Seeger C, Sohn JA. Targeting hepatitis B virus with CRISPR/Cas9. Mol Ther Nucleic Acids. 2014; 3. DOI: 10.1038/mtna.2014.68

Kostyushev D, Brezgin S, Kostyusheva A, Zarifyan D, Goptar I, Chulanov V. Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus. Cell Mol Life Sci. 2019; 76:1779–94. DOI: 10.1007/s00018-019-03021-8

Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 2018; 25:1234–57. DOI: 10.1080/10717544.2018.1474964

Cheng X, Fan S, Wen C, Du X. CRISPR/Cas9 for cancer treatment: technology, clinical applications and challenges. Brief Funct Genomics. 2020; 19:209–14. DOI: 10.1093/bfgp/elaa001

Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31:822–6. DOI: 10.1038/nbt.2623

Chen X, Gonçalves MAFV. Engineered viruses as genome editing devices. Mol Ther. 2016;24:447-457. DOI: 10.1038/mt.2015.164

Kay MA. State-of-the-art gene-based therapies: the road ahead. Nat Rev Genet [Internet]. 2011; 12:316–28. DOI: 10.1038/nrg2971

Naso MF, Tomkowicz B, Perry WL III, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017; 31:317–34. DOI: 10.1007/s40259-017-0234-5

Tyagi S, Kumar R, Das A, Won SY, Shukla P. CRISPR-Cas9 system: A genome-editing tool with endless possibilities. J Biotechnol. 2020; 319:36–53. DOI: 10.1016/j.jbiotec.2020.05.008

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Publicado

2023-09-29

Cómo citar

Vaglio-Cedeño, C., Rodríguez, E. J., & Morales, F. (2023). Aplicaciones clínicas de la herramienta CRISPR-Cas. Acta Médica Costarricense, 65(3), 1–11. https://doi.org/10.51481/amc.v65i3.1268