Current Gene TherapyCurrent Gene TherapyCurrent Gene Therapy1566-52321875-5631Bentham Science64405410.2174/0115665232279528240115075352Life SciencesResearch ArticlePrecision Genome Editing Techniques in Gene Therapy: Current State and Future ProspectsSinghKuldeepinfo@benthamscience.netBhushanBharatinfo@benthamscience.netKumarSunilinfo@benthamscience.netSinghSupriyainfo@benthamscience.netMacadangdangRomuloinfo@benthamscience.netPandeyEktainfo@benthamscience.netVarmaAjitinfo@benthamscience.netKumarShivendrainfo@benthamscience.netDepartment of Pharmacology, Rajiv Academy for PharmacyDepartment of Pharmacology, Institute of Pharmaceutical Research,, GLA UniversityDepartment of Pharmacology, P.K. UniversityDepartment of Pharmaceutics, Babu Banarasi Das Northern India Institute of Technology LucknowDepartment of Nursing, College of Allied Health,, National UniversityDepartment of Chemistry, Bundelkhand Institute of Engineering and TechnologyDepartment of Pharmaceutics, Rama University0105202424537739407012025Copyright ©; 2024, Bentham Science Publishers2024Bentham Science Publishershttps://journals.eco-vector.com/1566-5232/article/view/644054

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPR-- Cas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

Precision genome editinggene therapyCRISPR-Cas9genetic disorderstherapeutic interventionsbase editingprime editing.Chavez M, Chen X, Finn PB, Qi LS. Advances in CRISPR therapeutics. Nat Rev Nephrol 2023; 19(1): 9-22. doi: 10.1038/s41581-022-00636-2 PMID: 36280707Wang X, Ma C, Labrada R, et al. Recent advances in lentiviral vectors for gene therapy. Sci China Life Sci 2021; 64(11): 1842-57. doi: 10.1007/s11427-021-1952-5 PMID: 34708326Temin HM. Mixed infection with two types of Rous sarcoma virus. Virology 1961; 13(2): 158-63. doi: 10.1016/0042-6822(61)90049-6 PMID: 13775833Sambrook J, Westphal H, Srinivasan PR, Dulbecco R. The integrated state of viral DNA in SV40-transformed cells. Proc Natl Acad Sci USA 1968; 60(4): 1288-95. doi: 10.1073/pnas.60.4.1288 PMID: 4299943Tatum EL. Molecular biology, nucleic acids, and the future of medicine. Perspect Biol Med 1966; 10(1): 19-32. doi: 10.1353/pbm.1966.0027 PMID: 6002665Mercola KE, Bar-Eli M, Stang HD, Slamon DJ, Cline MJ. Insertion of new genetic information into bone marrow cells of mice: Comparison of two selectable genes. Ann N Y Acad Sci 1982; 397(1): 272-80. doi: 10.1111/j.1749-6632.1982.tb43434.x PMID: 6297353Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346(6213): 1258096. doi: 10.1126/science.1258096 PMID: 25430774Kang L, Jin S, Wang J, et al. AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges. J Control Release 2023; 355: 458-73. doi: 10.1016/j.jconrel.2023.01.067 PMID: 36736907Morange M. What history tells us XXXVII. CRISPR-Cas: The discovery of an immune system in prokaryotes. J Biosci 2015; 40(2): 221-3. doi: 10.1007/s12038-015-9532-6 PMID: 25963251Varshavsky A. Discovering the RNA double helix and hybridization. Cell 2006; 127(7): 1295-7. doi: 10.1016/j.cell.2006.12.008 PMID: 17190591Faruqi AF, Seidman MM, Segal DJ, Carroll D, Glazer PM. Recombination induced by triple-helix-targeted DNA damage in mammalian cells. Mol Cell Biol 1996; 16(12): 6820-8. doi: 10.1128/MCB.16.12.6820 PMID: 8943337Jiang F, Doudna JA. CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys 2017; 46(1): 505-29. doi: 10.1146/annurev-biophys-062215-010822 PMID: 28375731Bandyopadhyay A, Kancharla N, Javalkote VS, Dasgupta S, Brutnell TP. CRISPR-Cas12a (Cpf1): A versatile tool in the plant genome editing tool box for agricultural advancement. Front Plant Sci 2020; 11: 584151. doi: 10.3389/fpls.2020.584151 PMID: 33214794Ali Z, Mahas A, Mahfouz M. CRISPR/Cas13 as a tool for RNA interference. Trends Plant Sci 2018; 23(5): 374-8. doi: 10.1016/j.tplants.2018.03.003 PMID: 29605099Sun N, Zhao H. Transcription activator-like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing. Biotechnol Bioeng 2013; 110(7): 1811-21. doi: 10.1002/bit.24890 PMID: 23508559Cathomen T, Keith Joung J. Zinc-finger nucleases: The next generation emerges. Mol Ther 2008; 16(7): 1200-7. doi: 10.1038/mt.2008.114Porto EM, Komor AC, Slaymaker IM, Yeo GW. Base editing: Advances and therapeutic opportunities. Nat Rev Drug Discov 2020; 19(12): 839-59. doi: 10.1038/s41573-020-0084-6 PMID: 33077937Scholefield J, Harrison PT. Prime editing – An update on the field. Gene Ther 2021; 28(7-8): 396-401. doi: 10.1038/s41434-021-00263-9 PMID: 34031549Saeed S, Usman B, Shim SH, et al. CRISPR/Cas-mediated editing of cis-regulatory elements for crop improvement. Plant Sci 2022; 324: 111435. doi: 10.1016/j.plantsci.2022.111435 PMID: 36031021Nakamura M, Gao Y, Dominguez AA, Qi LS. CRISPR technologies for precise epigenome editing. Nat Cell Biol 2021; 23(1): 11-22. doi: 10.1038/s41556-020-00620-7 PMID: 33420494Lienert F, Lohmueller JJ, Garg A, Silver PA. Synthetic biology in mammalian cells: Next generation research tools and therapeutics. Nat Rev Mol Cell Biol 2014; 15(2): 95-107. doi: 10.1038/nrm3738 PMID: 24434884Ishino 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(12): 5429-33. doi: 10.1128/jb.169.12.5429-5433.1987 PMID: 3316184Jansen R, Embden JDA, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75. doi: 10.1046/j.1365-2958.2002.02839.x PMID: 11952905Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60(2): 174-82. doi: 10.1007/s00239-004-0046-3 PMID: 15791728Garneau JE, Dupuis MÈ, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010; 468(7320): 67-71. doi: 10.1038/nature09523 PMID: 21048762Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol 2011; 9(6): 467-77. doi: 10.1038/nrmicro2577 PMID: 21552286Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 2010; 64(1): 475-93. doi: 10.1146/annurev.micro.112408.134123 PMID: 20528693Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: Versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45(1): 273-97. doi: 10.1146/annurev-genet-110410-132430 PMID: 22060043Brouns SJJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321(5891): 960-4. doi: 10.1126/science.1159689 PMID: 18703739Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322(5909): 1843-5. doi: 10.1126/science.1165771 PMID: 19095942Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 2011; 39(21): 9275-82. doi: 10.1093/nar/gkr606 PMID: 21813460Magadán AH, Dupuis MÈ, Villion M, Moineau S. Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS One 2012; 7(7): e40913. doi: 10.1371/journal.pone.0040913 PMID: 22911717Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012; 109(39): E2579-86. doi: 10.1073/pnas.1208507109 PMID: 22949671Jinek 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(6096): 816-21. doi: 10.1126/science.1225829 PMID: 22745249Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339(6121): 819-23. doi: 10.1126/science.1231143 PMID: 23287718Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: A review of the challenges and approaches. Drug Deliv 2018; 25(1): 1234-57. doi: 10.1080/10717544.2018.1474964 PMID: 29801422Zhang Y, Heidrich N, Ampattu BJ, et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell 2013; 50(4): 488-503. doi: 10.1016/j.molcel.2013.05.001 PMID: 23706818Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013; 31(3): 230-2. doi: 10.1038/nbt.2507 PMID: 23360966Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013; 31(3): 227-9. doi: 10.1038/nbt.2501 PMID: 23360964Shen B, Zhang J, Wu H, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 2013; 23(5): 720-3. doi: 10.1038/cr.2013.46 PMID: 23545779Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153(4): 910-8. doi: 10.1016/j.cell.2013.04.025 PMID: 23643243Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013; 154(6): 1380-9. doi: 10.1016/j.cell.2013.08.021 PMID: 23992846Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152(5): 1173-83. doi: 10.1016/j.cell.2013.02.022 PMID: 23452860Doudna JA, Charpentier E. Genome editing. The new front genome eng CRISPR-Cas. Science 2014; 346(6213): 1258096.Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157(6): 1262-78. doi: 10.1016/j.cell.2014.05.010 PMID: 24906146Kim YG, Shi Y, Berg JM, Chandrasegaran S. Site-specific cleavage of DNA–RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene 1997; 203(1): 43-9. doi: 10.1016/S0378-1119(97)00489-7 PMID: 9426005Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 1996; 93(3): 1156-60. doi: 10.1073/pnas.93.3.1156 PMID: 8577732Bibikova M, Carroll D, Segal DJ, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 2001; 21(1): 289-97. doi: 10.1128/MCB.21.1.289-297.2001 PMID: 11113203Bibikova M, Golic M, Golic KG, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 2002; 161(3): 1169-75. doi: 10.1093/genetics/161.3.1169 PMID: 12136019Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science 2003; 300(5620): 764. doi: 10.1126/science.1079512 PMID: 12730594Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 2009; 325(5939): 433. doi: 10.1126/science.1172447 PMID: 19628861Tong C, Li P, Wu NL, Yan Y, Ying QL. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 2010; 467(7312): 211-3. doi: 10.1038/nature09368 PMID: 20703227Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 2010; 186(2): 451-9. doi: 10.1534/genetics.110.117002 PMID: 20628038Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 2010; 11(9): 636-46. doi: 10.1038/nrg2842 PMID: 20717154Gao H, Wu X, Chai J, Han Z. Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region. Cell Res 2012; 22(12): 1716-20. doi: 10.1038/cr.2012.156 PMID: 23147789Szurek B, Rossier O, Hause G, Bonas U. Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol Microbiol 2002; 46(1): 13-23. doi: 10.1046/j.1365-2958.2002.03139.x PMID: 12366827Yuan M, Ke Y, Huang R, et al. A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria. eLife 2016; 5: e19605. doi: 10.7554/eLife.19605 PMID: 27472897Zhu W, Yang B, Chittoor JM, Johnson LB, White FF. AvrXa10 contains an acidic transcriptional activation domain in the functionally conserved C terminus. Mol Plant Microbe Interact 1998; 11(8): 824-32. doi: 10.1094/MPMI.1998.11.8.824 PMID: 9675896Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009; 326(5959): 1509-12. doi: 10.1126/science.1178811 PMID: 19933107Yang J, Zhang Y, Yuan P, et al. Complete decoding of TAL effectors for DNA recognition. Cell Res 2014; 24(5): 628-31. doi: 10.1038/cr.2014.19 PMID: 24513857Miller JC, Zhang L, Xia DF, et al. Improved specificity of TALE-based genome editing using an expanded RVD repertoire. Nat Methods 2015; 12(5): 465-71. doi: 10.1038/nmeth.3330 PMID: 25799440Deng D, Yan C, Pan X, et al. Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 2012; 335(6069): 720-3. doi: 10.1126/science.1215670 PMID: 22223738Mak ANS, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL. The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 2012; 335(6069): 716-9. doi: 10.1126/science.1216211 PMID: 22223736Lamb BM, Mercer AC, Barbas CF III. Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases. Nucleic Acids Res 2013; 41(21): 9779-85. doi: 10.1093/nar/gkt754 PMID: 23980031Streubel J, Blücher C, Landgraf A, Boch J. TAL effector RVD specificities and efficiencies. Nat Biotechnol 2012; 30(7): 593-5. doi: 10.1038/nbt.2304 PMID: 22781676Richter A, Streubel J, Blücher C, et al. A TAL effector repeat architecture for frameshift binding. Nat Commun 2014; 5(1): 3447. doi: 10.1038/ncomms4447 PMID: 24614980Yang LH, Briggs AW, Chew WL, Mali P, Guell M, Aach J, et al. Engineering and optimising deaminase fusions for genome editing (vol 7, 13330, 2016). Nat Commun 2017; 8: 16169. doi: 10.1038/ncomms16169 PMID: 28991237Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 2017; 35(4): 371-6. doi: 10.1038/nbt.3803 PMID: 28191901Li Z, Abraham BJ, Berezovskaya A, et al. APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL. Leukemia 2017; 31(10): 2057-64. doi: 10.1038/leu.2017.75 PMID: 28260788Yang L, Briggs AW, Chew WL, et al. Engineering and optimising deaminase fusions for genome editing. Nat Commun 2016; 7(1): 13330. doi: 10.1038/ncomms13330 PMID: 27804970Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 2017; 551(7681): 464-71. advance online publication doi: 10.1038/nature24644 PMID: 29160308Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019; 576(7785): 149-57. doi: 10.1038/s41586-019-1711-4 PMID: 31634902Benne R, Van Den Burg J, Brakenhoff JPJ, Sloof P, Van Boom JH, Tromp MC. Major transcript of the frameshifted coxll gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 1986; 46(6): 819-26. doi: 10.1016/0092-8674(86)90063-2 PMID: 3019552Grosjean H, Benne R. Modification and editing of RNA. Washington, DC: ASM Press 1998; pp. XI-XIII. doi: 10.1128/9781555818296Lane BG. Historical perspectives on RNA nucleoside modifications. In: Modification and editing of RNA. Washington, DC: ASM Press 1998; pp. 1-20.Ylä-Herttuala S. Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union. Mol Ther 2012; 20(10): 1831-2. doi: 10.1038/mt.2012.194 PMID: 23023051Gaudet D, Méthot J, Kastelein J. Gene therapy for lipoprotein lipase deficiency. Curr Opin Lipidol 2012; 23(4): 310-20. doi: 10.1097/MOL.0b013e3283555a7e PMID: 22691709Stroes ES, Nierman MC, Meulenberg JJ, et al. Intramuscular administration of AAV1-lipoprotein lipase S447X lowers triglycerides in lipoprotein lipase-deficient patients. Arterioscler Thromb Vasc Biol 2008; 28(12): 2303-4. doi: 10.1161/ATVBAHA.108.175620 PMID: 18802015Ferreira V, Petry H, Salmon F. Immune responses to AAV-vectors, the glybera example from bench to bedside. Front Immunol 2014; 5: 82. a doi: 10.3389/fimmu.2014.00082 PMID: 24624131Malina A, Mills JR, Cencic R, et al. Repurposing CRISPR/Cas9 for in situ functional assays. Genes Dev 2013; 27(23): 2602-14. doi: 10.1101/gad.227132.113 PMID: 24298059Chen C, Liu Y, Rappaport AR, et al. MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia. Cancer Cell 2014; 25(5): 652-65. doi: 10.1016/j.ccr.2014.03.016 PMID: 24794707Xue W, Chen S, Yin H, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 2014; 514(7522): 380-4. doi: 10.1038/nature13589 PMID: 25119044Yahata T, Mizoguchi M, Kimura A, et al. Programmed cell death ligand 1 D isruption by clustered regularly interspaced short palindromic repeats /Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression. Cancer Sci 2019; 110(4): 1279-92. doi: 10.1111/cas.13958 PMID: 30702189Deng H, Tan S, Gao X, et al. Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity. Acta Pharm Sin B 2020; 10(2): 358-73. doi: 10.1016/j.apsb.2019.07.004 PMID: 32082979Su S, Zou Z, Chen F, et al. CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer. OncoImmunology 2017; 6(1): e1249558. doi: 10.1080/2162402X.2016.1249558 PMID: 28197365Mahoney KM, Freeman GJ, McDermott DF. The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther 2015; 37(4): 764-82. doi: 10.1016/j.clinthera.2015.02.018 PMID: 25823918Choi BD, Yu X, Castano AP, et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. J Immunother Cancer 2019; 7(1): 304. doi: 10.1186/s40425-019-0806-7 PMID: 31727131Gao SP, Kiliti AJ, Zhang K, et al. AKT1 E17K inhibits cancer cell migration by abrogating β-catenin signaling. Mol Cancer Res 2021; 19(4): 573-84. doi: 10.1158/1541-7786.MCR-20-0623 PMID: 33303690Shi Y, Wang G, Cai X, et al. An overview of COVID-19. J Zhejiang Univ Sci B 2020; 21(5): 343-60. doi: 10.1631/jzus.B2000083 PMID: 32425000Kumar P, Malik YS, Ganesh B, et al. CRISPR-Cas system: An approach with potentials for COVID-19 diagnosis and therapeutics. Front Cell Infect Microbiol 2020; 10: 576875. doi: 10.3389/fcimb.2020.576875 PMID: 33251158Chertow DS. Next-generation diagnostics with CRISPR. Science 2018; 360(6387): 381-2. doi: 10.1126/science.aat4982 PMID: 29700254Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 2017; 356(6336): 438-42. doi: 10.1126/science.aam9321 PMID: 28408723Wang X, Zhong M, Liu Y, et al. Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER. Sci Bull 2020; 65(17): 1436-9. b doi: 10.1016/j.scib.2020.04.041 PMID: 32373393Kanitchinda S, Srisala J, Suebsing R, Prachumwat A, Chaijarasphong T. CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei. Biotechnol Rep 2020; 27: e00485. doi: 10.1016/j.btre.2020.e00485 PMID: 32577410Li Z, Wei J, Di D, et al. Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay. Acta Biochim Biophys Sin 2020; 52(12): 1413-9. doi: 10.1093/abbs/gmaa135 PMID: 33201182Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: Nucleic acid detection with CRISPR nucleases. Nat Protoc 2019; 14(10): 2986-3012. doi: 10.1038/s41596-019-0210-2 PMID: 31548639Mustafa MI, Makhawi AM. Sherlock and DETECTR: CRISPR-Cas systems as potential rapid diagnostic tools for emerging infectious diseases. J Clin Microbiol 2021; 59(3): e00745-20. doi: 10.1128/JCM.00745-20 PMID: 33148705Brandsma E, Verhagen HJMP, van de Laar TJW, Claas ECJ, Cornelissen M, van den Akker E. Rapid, sensitive, and specific severe acute respiratory syndrome coronavirus 2 detection: A multicenter comparison between standard quantitative reverse-transcriptase polymerase chain reaction and CRISPR-based DETECTR. J Infect Dis 2021; 223(2): 206-13. doi: 10.1093/infdis/jiaa641 PMID: 33535237Wang M, Zhang R, Li J. CRISPR/cas systems redefine nucleic acid detection: Principles and methods. Biosens Bioelectron 2020; 165: 112430. doi: 10.1016/j.bios.2020.112430 PMID: 32729545Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun 2020; 11(1): 4711. doi: 10.1038/s41467-020-18575-6 PMID: 32948757Javalkote VS, Kancharla N, Bhadra B, et al. CRISPR-based assays for rapid detection of SARS-CoV-2. Methods 2022; 203: 594-603. doi: 10.1016/j.ymeth.2020.10.003 PMID: 33045362Ali Z, Aman R, Mahas A, et al. iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res 2020; 288: 198129. doi: 10.1016/j.virusres.2020.198129 PMID: 32822689Quan J, Langelier C, Kuchta A, et al. FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. Nucleic Acids Res 2019; 47(14): e83. doi: 10.1093/nar/gkz418 PMID: 31114866Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science 2018; 360(6387): 444-8. doi: 10.1126/science.aas8836 PMID: 29700266Ai JW, Zhou X, Xu T, et al. CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis. Emerg Microbes Infect 2019; 8(1): 1361-9. doi: 10.1080/22221751.2019.1664939 PMID: 31522608Tian X, Gu T, Patel S, Bode AM, Lee MH, Dong Z. CRISPR/Cas9 – An evolving biological tool kit for cancer biology and oncology. NPJ Precis Oncol 2019; 3(1): 8. doi: 10.1038/s41698-019-0080-7 PMID: 30911676Xu L, Wang J, Liu Y, et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N Engl J Med 2019; 381(13): 1240-7. doi: 10.1056/NEJMoa1817426 PMID: 31509667Stadtmauer EA, Fraietta JA, Davis MM, et al. CRISPR-engineered T cells in patients with refractory cancer. Science 2020; 367(6481): eaba7365. doi: 10.1126/science.aba7365 PMID: 32029687Lu Y, Xue J, Deng T, et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med 2020; 26(5): 732-40. doi: 10.1038/s41591-020-0840-5 PMID: 32341578Cyranoski D, Ledford H. Genome-edited baby claim provokes international outcry. Nature 2018; 563(7733): 607-8. doi: 10.1038/d41586-018-07545-0 PMID: 30482929Savulescu J, Singer P. An ethical pathway for gene editing. Bioethics 2019; 33(2): 221-2. doi: 10.1111/bioe.12570 PMID: 30695116Nuffield Council on Bioethics Genome editing and human reproduction: social and ethical issues. Nuffield Council on Bioethics 2018.Gyngell C, Bowman-Smart H, Savulescu J. Moral reasons to edit the human genome: picking up from the Nuffield report. J Med Ethics 2019; 45(8): 514-23. doi: 10.1136/medethics-2018-105084 PMID: 30679191Cavaliere G. The ethics of human genome editing. WHO expert advisory committee on developing global standards for governance and oversight of human genome editing 2019. Available from: https://www.who.int/ethics/topics/human-genome-editing/WHO-Commissioned-Ethics-paper-March19. pdf accessed Jan 25 2021.Mahajan R. Onasemnogene abeparvovec for spinal muscular atrophy: The costlier drug ever. Int J Appl Basic Med Res 2019; 9(3): 127-8. doi: 10.4103/ijabmr.IJABMR_190_19 PMID: 31392173Seimetz D, Heller K, Richter J. Approval of first CAR-Ts: Have we solved all hurdles for ATMPs? Cell Med 2019; 11 doi: 10.1177/2155179018822781 PMID: 32634192Padhy SK, Takkar B, Narayanan R, Venkatesh P, Jalali S. Voretigene neparvovec and gene therapy for Leber’s congenital amaurosis: review of evidence to date. Appl Clin Genet 2020; 13: 179-208. doi: 10.2147/TACG.S230720 PMID: 33268999Gruber K. Europe gives gene therapy the green light. Lancet 2012; 380(9855): e10. doi: 10.1016/S0140-6736(12)61992-8 PMID: 23166921Zhang WW, Li L, Li D, et al. The first approved gene therapy product for cancer Ad-p53 (gendicine): 12 years in the clinic. Hum Gene Ther 2018; 29(2): 160-79. doi: 10.1089/hum.2017.218 PMID: 29338444Liang M. Oncorine, the world first oncolytic virus medicine and its update in China. Curr Cancer Drug Targets 2018; 18(2): 171-6. doi: 10.2174/1568009618666171129221503 PMID: 29189159Frangoul H, Altshuler D, Cappellini MD, et al. CRISPR-Cas9 gene editing for sickle cell disease and beta-thalassemia. N Engl J Med 2021; 384(3): 252-60. doi: 10.1056/NEJMoa2031054 PMID: 33283989Gillmore JD, Gane E, Taubel J, et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med 2021; 385(6): 493-502. doi: 10.1056/NEJMoa2107454 PMID: 34215024Ma CC, Wang ZL, Xu T, He ZY, Wei YQ. The approved gene therapy drugs worldwide: From 1998 to 2019. Biotechnol Adv 2020; 40: 107502. doi: 10.1016/j.biotechadv.2019.107502 PMID: 31887345Shahryari A, Saghaeian Jazi M, Mohammadi S, et al. Development and clinical translation of approved gene therapy products for genetic disorders. Front Genet 2019; 10: 868. doi: 10.3389/fgene.2019.00868 PMID: 31608113Zhang X, Chen L, Zhu B, et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat Cell Biol 2020; 22(6): 740-50. doi: 10.1038/s41556-020-0518-8 PMID: 32393889Koblan LW, Doman JL, Wilson C, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 2018; 36(9): 843-6. doi: 10.1038/nbt.4172 PMID: 29813047Zhao D, Li J, Li S, et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 2021; 39(1): 35-40. doi: 10.1038/s41587-020-0592-2 PMID: 32690970Ousterout DG, Perez-Pinera P, Thakore PI, et al. Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol Ther 2013; 21(9): 1718-26. doi: 10.1038/mt.2013.111 PMID: 23732986Osborn MJ, Starker CG, McElroy AN, et al. TALEN-based gene correction for epidermolysis bullosa. Mol Ther 2013; 21(6): 1151-9. doi: 10.1038/mt.2013.56 PMID: 23546300Genovese P, Schiroli G, Escobar G, et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 2014; 510(7504): 235-40. doi: 10.1038/nature13420 PMID: 24870228Crane AM, Kramer P, Bui JH, et al. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells. Stem Cell Reports 2015; 4(4): 569-77. doi: 10.1016/j.stemcr.2015.02.005 PMID: 25772471Corvol H, Thompson KE, Tabary O, le Rouzic P, Guillot L. Translating the genetics of cystic fibrosis to personalized medicine. Transl Res 2016; 168: 40-9. doi: 10.1016/j.trsl.2015.04.008 PMID: 25940043Hoegger MJ, Fischer AJ, McMenimen JD, et al. Impaired mucus detachment disrupts mucociliary transport in a piglet model of cystic fibrosis. Science 2014; 345(6198): 818-22. doi: 10.1126/science.1255825 PMID: 25124441Sallenave JM. Phagocytic and signaling innate immune receptors: Are they dysregulated in cystic fibrosis in the fight against Pseudomonas aeruginosa? Int J Biochem Cell Biol 2014; 52: 103-7. doi: 10.1016/j.biocel.2014.01.013 PMID: 24508137Wong AP, Bear CE, Chin S, et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 2012; 30(9): 876-82. doi: 10.1038/nbt.2328 PMID: 22922672Nishitani C, Hirai N, Komori S, et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep 2016; 6(1): 31481. doi: 10.1038/srep31481 PMID: 27530958Wang Z, Wang S, Li D, et al. Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol J 2018; 16(8): 1424-33. doi: 10.1111/pbi.12884 PMID: 29331077Breitler JC, Dechamp E, Campa C, et al. CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of Coffea canephora. Plant Cell Tissue Organ Cult 2018; 134(3): 383-94. doi: 10.1007/s11240-018-1429-2Odipio J, Alicai T, Ingelbrecht I, Nusinow DA, Bart R, Taylor NJ. Efficient CRISPR/Cas9 genome editing of phytoene desaturase in cassava. Front Plant Sci 2017; 8: 1780. doi: 10.3389/fpls.2017.01780 PMID: 29093724Osakabe Y, Liang Z, Ren C, et al. CRISPR–Cas9-mediated genome editing in apple and grapevine. Nat Protoc 2018; 13(12): 2844-63. doi: 10.1038/s41596-018-0067-9 PMID: 30390050van Regenmortel MH, Mahy BW. Desk encyclopedia of plant and fungal virology. San Diego: Elsevier 2009.Ali Z, Ali S, Tashkandi M, Zaidi SSA, Mahfouz MM. CRISPR/Cas9-mediated immunity to geminiviruses: Differential interference and evasion. Sci Rep 2016; 6(1): 26912. doi: 10.1038/srep26912 PMID: 27225592Tashkandi M, Ali Z, Aljedaani F, Shami A, Mahfouz MM. Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato. Plant Signal Behav 2018; 13(10): e1525996. doi: 10.1080/15592324.2018.1525996 PMID: 30289378Ishii T. Germline genome-editing research and its socioethical implications Trends Mol Med 2015; 21(8): 473-81. doi: 10.1016/j.molmed.2015.05.006Janssens AC. Designing babies through gene editing: science or science fction? Genet Med 2016; 18(12): 1186-7. doi: 10.1038/gim.2016.28Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, et al. Biotechnology. A prudent path forward for genomic engineering and germline gene modifcation. Science 2015; 348(6230): 36-8. doi: 10.1126/science.aab1028Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J. Don’t edit the human germ line. Nature 2015; 519(7544): 410-1. doi: 10.1038/519410a