CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases
Authors: Garima Sharma, Ashish Ranjan Sharma, Manojit Bhattacharya, Sang-Soo Lee, Chiranjib Chakraborty
Journal: Molecular Therapy (2020)

At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have traveled a long way from gene modifications in primary cells to genetic modifications in animals. The targeted genetic modification may result in the modulation (i.e., either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) is a promising genome-editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome-editing techniques, CRISPR-Cas9 is simple, efficient, and very specific. This enabled CRISPR-Cas9 genome-editing technology to enter into clinical trials against cancer. Besides therapeutic potential, the CRISPR-Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discusses the origin of CRISPR-Cas9 systems and their therapeutic potential against various genetic disorders, including cancer, allergy, immunological disorders, Duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders, and viral infection. Finally, we discuss the different challenges, safety concerns, and strategies that can be applied to overcome the obstacles during CRISPR-Cas9-mediated therapeutic approaches. At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have traveled a long way from gene modifications in primary cells to genetic modifications in animals. The targeted genetic modification may result in the modulation (i.e., either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) is a promising genome-editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome-editing techniques, CRISPR-Cas9 is simple, efficient, and very specific. This enabled CRISPR-Cas9 genome-editing technology to enter into clinical trials against cancer. Besides therapeutic potential, the CRISPR-Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discusses the origin of CRISPR-Cas9 systems and their therapeutic potential against various genetic disorders, including cancer, allergy, immunological disorders, Duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders, and viral infection. Finally, we discuss the different challenges, safety concerns, and strategies that can be applied to overcome the obstacles during CRISPR-Cas9-mediated therapeutic approaches. Clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a novel and competent RNA-guided endonuclease-based genome-editing technique1Young C.S. Pyle A.D. Spencer M.J. CRISPR for neuromuscular disorders: gene editing and beyond.Physiology (Bethesda). 2019; 34: 341-353PubMed Google Scholar that is adapted from the naturally occurring bacterial immune system. The CRISPR-Cas9 technique is easily scalable and very cost-effective, and thus it can be applied for a wide range of directed genome editing.2Chakraborty C. Teoh S.L. Das S. The smart programmable CRISPR technology: a next generation genome editing tool for investigators.Curr. Drug Targets. 2017; 18: 1653-1663Crossref PubMed Scopus (3) Google Scholar, 3Hsu P.D. Lander E.S. Zhang F. 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Genome-editing technology has gained attention as a therapy against human diseases. However, its success has been previously challenged by unpredictable outcomes. In early gene therapy trials, 5 children, out of 20 participants from different studies, suffering from the severe combined immunodeficiency (SCID) X-1 condition developed T cell leukemia due to non-specific insertion of a correcting gene near to tumor-promoting genes, which resulted in transcriptional activation.21Check E. A tragic setback.Nature. 2002; 420: 116-118Crossref PubMed Scopus (245) Google Scholar, 22Kaiser J. Gene therapy. Seeking the cause of induced leukemias in X-SCID trial.Science. 2003; 299: 495Crossref PubMed Scopus (106) Google Scholar, 23Lino C.A. Harper J.C. Carney J.P. Timlin J.A. Delivering CRISPR: a review of the challenges and approaches.Drug Deliv. 2018; 25: 1234-1257Crossref PubMed Scopus (307) Google Scholar, 24Gaspar H.B. Cooray S. Gilmour K.C. Parsley K.L. Adams S. Howe S.J. Al Ghonaium A. 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Delivering CRISPR: a review of the challenges and approaches.Drug Deliv. 2018; 25: 1234-1257Crossref PubMed Scopus (307) Google Scholar Lately, more advanced gene therapy technologies, such as zinc finger nucleases (ZFNs),27Bibikova M. Beumer K. Trautman J.K. Carroll D. Enhancing gene targeting with designed zinc finger nucleases.Science. 2003; 300: 764Crossref PubMed Scopus (628) Google Scholar transcription activator-like effector nucleases (TALENs),28Christian M. Cermak T. Doyle E.L. Schmidt C. Zhang F. Hummel A. Bogdanove A.J. Voytas D.F. Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1167) Google Scholar and CRISPR-Cas9,29Jansen R. Embden J.D. Gaastra W. Schouls L.M. Identification of genes that are associated with DNA repeats in prokaryotes.Mol. Microbiol. 2002; 43: 1565-1575Crossref PubMed Scopus (991) Google Scholar were developed, which were capable of site-specific gene editing. ZFNs are a combination of a non-specific FokI cleavage domain and zinc finger proteins (ZFPs). In eukaryotes, ZFPs are associated with protein-protein interaction and regulation of DNA transcription.30Wolfe S.A. Nekludova L. Pabo C.O. DNA recognition by Cys2His2 zinc finger proteins.Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 183-212Crossref PubMed Scopus (732) Google Scholar For gene editing, paired ZFNs, one downstream and one upstream of the target site, are used to generate DSBs.31Doyon Y. Vo T.D. Mendel M.C. Greenberg S.G. Wang J. Xia D.F. Miller J.C. Urnov F.D. Gregory P.D. Holmes M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat. Methods. 2011; 8: 74-79Crossref PubMed Scopus (302) Google Scholar ZFNs, however, can only recognize nucleotide triplets in the DNA, thus restricting the number of site selections due to limited binding sites for ZFPs.32Ramirez C.L. Foley J.E. Wright D.A. Müller-Lerch F. Rahman S.H. Cornu T.I. 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Schmidt C. Zhang F. Hummel A. Bogdanove A.J. Voytas D.F. Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1167) Google Scholar TALEs are the amino acid sequences that flank a DNA binding site. TALENs have a benefit over ZFNs, as they can recognize a single nucleotide and are more specific than ZFNs.35Joung J.K. Sander J.D. TALENs: a widely applicable technology for targeted genome editing.Nat. Rev. Mol. Cell Biol. 2013; 14: 49-55Crossref PubMed Scopus (917) Google Scholar Still, packaging and delivery of TALENs might be a challenge because they have a large size than ZFNs.23Lino C.A. Harper J.C. Carney J.P. Timlin J.A. Delivering CRISPR: a review of the challenges and approaches.Drug Deliv. 2018; 25: 1234-1257Crossref PubMed Scopus (307) Google Scholar CRISPR-Cas9 is latest gene-editing technique with various advantages over ZFNs and TALENs. First, CRISPR-Cas9 is more cost-effective than other gene-editing techniques.36Bhattacharya D. Marfo C.A. Li D. Lane M. Khokha M.K. CRISPR/Cas9: an inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.Dev. Biol. 2015; 408: 196-204Crossref PubMed Scopus (78) Google Scholar Second, the target specificity of CRISPR-Cas9 depends on the ribonucleotide complex, which is probably more specific to protein-based DNA bindings in ZFNs and TALENs. Moreover, it is easy to design sgRNA for a wide range of target DNAs. In addition, various modifications can be performed in the CRISPR-Cas9 cargo system, i.e., plasmid DNA encoding sgRNA and Cas9, the combination of sgRNA and Cas9 mRNA, and the combination of sgRNA and Cas9 protein. Other advantages of CRISPR-Cas9 include the possibility of direct genome modification in the embryo and introduction of more than one mutation at the same time.37Yang H. Wu J.-J. Tang T. Liu K.-D. Dai C. CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus.Sci. Rep. 2017; 7: 7489Crossref PubMed Scopus (87) Google Scholar In addition, base edition (i.e., the conversion of cytidine to uracil [C→T] or guanine to adenine [G→A]) that is mediated by the binding of CRISPR-Cas9 with cytidine deaminase is also possible.38Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (1658) Google Scholar These advancements indicate the possibility of safe and successful clinical application of CRISPR-Cas9 technology in the future. CRISPR-Cas9 research has been applied for the treatment of different human diseases (Table1), which are discussed below.Table 1CRISPR-Cas9 Research and Its Application for the Treatment of Different Human DiseasesDisease TypeDisease-Related Gene/ProteinRemarksReferencesCanceradditional sex combs like 1 (ASXL1)CRISPR-Cas9 was used to decrease leukemia cell growth in mouse xenografts39Valletta S. Dolatshad H. Bartenstein M. Yip B.H. Bello E. Gordon S. Yu Y. Shaw J. Roy S. Scifo L. et al.ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.Oncotarget. 2015; 6: 44061-44071Crossref PubMed Scopus (43) Google Scholarmyeloid cell leukemia 1 (MCL-1)CRISPR-Cas9 was used to delete MCL-1 in human BL cells and induce apoptosis in the BL cells40Aubrey B.J. Kelly G.L. Kueh A.J. Brennan M.S. O'Connor L. Milla L. Wilcox S. Tai L. Strasser A. Herold M.J. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.Cell Rep. 2015; 10: 1422-1432Abstract Full Text Full Text PDF PubMed Google Scholarcyclin-dependent kinase 11 (CDK11)CRISPR-Cas9 was used to silence CDK11 in osteosarcoma41Feng Y. Sassi S. Shen J.K. Yang X. Gao Y. Osaka E. Zhang J. Yang S. Yang C. Mankin H.J. et al.Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.J. Orthop. Res. 2015; 33: 199-207Crossref PubMed Scopus (47) Google ScholarSHC SH2-binding protein 1 (SHCBP1)SHCBP1 inhibits the proliferation of breast cancer through CRISPR-Cas942Feng W. Li H.C. Xu K. Chen Y.F. Pan L.Y. Mei Y. Cai H. Jiang Y.M. Chen T. Feng D.X. SHCBP1 is over-expressed in breast cancer and is important in the proliferation and apoptosis of the human malignant breast cancer cell line.Gene. 2016; 587: 91-97Crossref PubMed Scopus (41) Google ScholarKelch domain containing 4 (KLHDC4)CRISPR-Cas9 was used to knock out the KLHDC4 gene in a nasopharyngeal carcinoma cell line43Lian Y.-F. Yuan J. Cui Q. Feng Q.-S. Xu M. Bei J.-X. Zeng Y.X. Feng L. Upregulation of KLHDC4 predicts a poor prognosis in human nasopharyngeal carcinoma.PLoS ONE. 2016; 11: e0152820Crossref PubMed Scopus (18) Google Scholarepidermal growth factor receptor (EGFR)CRISPR-Cas9 was used for possible correction of acquired drug-resistant mutations in EGFR44Tang H. Shrager J.B. CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer: a personalized molecular surgical therapy.EMBO Mol. Med. 2016; 8: 83-85Crossref PubMed Scopus (0) Google ScholarAllergymelanoma cell adhesion molecule (MCAM/MUC18)CRISPR-Cas9 technology was used to knock out cell surface glycoprotein MUC18 in human primary nasal airway epithelial cells45Chu H.W. Rios C. Huang C. Wesolowska-Andersen A. Burchard E.G. O'Connor B.P. Fingerlin T.E. Nichols D. Reynolds S.D. Seibold M.A. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.Gene Ther. 2015; 22: 822-829Crossref PubMed Scopus (56) Google ScholarJanus kinase 3 (JAK3)CRISPR-Cas9 was used to re-establish the development of normal T cells in JAK3-deficient cells46Malaviya R. Laskin D.L. Malaviya R. Janus kinase-3 dependent inflammatory responses in allergic asthma.Int. Immunopharmacol. 2010; 10: 829-836Crossref PubMed Scopus (11) Google ScholarDuchenne muscular dystrophydystrophinCRISPR-Cas9 was used to fix DMD gene mutation for the DMD disorder47Long C. McAnally J.R. Shelton J.M. Mireault A.A. Bassel-Duby R. Olson E.N. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.Science. 2014; 345: 1184-1188Crossref PubMed Scopus (434) Google ScholarCardiovascular diseasesproprotein convertase subtilisin/kexin type 9 (PCSK9)CRISPR-Cas9 was used to correct the PCSK9 gene in an atherosclerosis mouse model48Seidah N.G. Proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors in the treatment of hypercholesterolemia and other pathologies.Curr. Pharm. Des. 2013; 19: 3161-3172Crossref PubMed Google ScholarHuntington diseasehuntingtin (HTT) geneCRISPR-Cas9 was used to suppress the mHTT gene selectively in a mouse model49Yang S. Chang R. Yang H. Zhao T. Hong Y. Kong H.E. Sun X. Qin Z. Jin P. Li S. Li X.J. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington's disease.J. Clin. Invest. 2017; 127: 2719-2724Crossref PubMed Scopus (171) Google ScholarAlzheimer's diseasepresenilin 1 (PSEN1) and presenilin 2 (PSEN2) genesCRISPR-Cas9 was used to correct ancestral mutations in AD related to the PSEN gene50Pires C. Schmid B. Petræus C. Poon A. Nimsanor N. Nielsen T.T. Waldemar G. Hjermind L.E. Nielsen J.E. Hyttel P. Freude K.K. Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1.Stem Cell Res. (Amst.). 2016; 17: 285-288Crossref PubMed Scopus (15) Google Scholar,51Rohn T.T. Kim N. Isho N.F. Mack J.M. The potential of CRISPR/Cas9 gene editing as a treatment strategy for Alzheimer's disease.J. Alzheimers Dis. Parkinsonism. 2018; 8: 8Crossref Google ScholarMetabolic liver diseasePahenu2CRISPR-Cas9 was used to correct the Pahenu2 gene in metabolic liver disease52Villiger L. Grisch-Chan H.M. Lindsay H. Ringnalda F. Pogliano C.B. Allegri G. Fingerhut R. Häberle J. Matos J. Robinson M.D. et al.Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.Nat. Med. 2018; 24: 1519-1525Crossref PubMed Scopus (139) Google ScholarFanconi anemia17 Fanconi anemia (FA)CRISPR-Cas9 was used to correct Fanconi anemia53Osborn M.J. Gabriel R. Webber B.R. DeFeo A.P. McElroy A.N. Jarjour J. Starker C.G. Wagner J.E. Joung J.K. Voytas D.F. et al.Fanconi anemia gene editing by the CRISPR/Cas9 system.Hum. Gene Ther. 2015; 26: 114-126Crossref PubMed Scopus (80) Google ScholarHereditary tyrosinemiafumarylacetoacetase (Fah)CRISPR-Cas9 was used to correct the Fah mutation in mouse models54Shao Y. Wang L. Guo N. Wang S. Yang L. Li Y. Wang M. Yin S. Han H. Zeng L. et al.Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.J. Biol. Chem. 2018; 293: 6883-6892Abstract Full Text Full Text PDF PubMed Scopus (24) Google ScholarSickle cell anemiaβ-globin geneCRISPR-Cas9 was used to treat sickle cell disease patient blood55Wen J. Tao W. 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Sci. 2017; 58: BIO34-BIO41Crossref PubMed Scopus (0) Google ScholarHuman immunodeficiency virus (HIV)long terminal repeats (LTRs) in HIVCRISPR-Cas9 was used as a tool to mutate LTRs of HIV-1 DNA60Ebina H. Misawa N. Kanemura Y. Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus.Sci. Rep. 2013; 3: 2510Crossref PubMed Scopus (345) Google ScholarHepatitis B virus (HBV)covalently closed circular DNAs (cccDNAs) in HBVCRISPR-Cas9 was used to target cccDNAs of HBV61Dong C. Qu L. Wang H. Wei L. Dong Y. Xiong S. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication.Antiviral Res. 2015; 118: 110-117Crossref PubMed Scopus (154) Google ScholarHuman papilloma virus (HPV)HPVE6 geneCRISPR-Cas9 was used to target HPVE6 for cancer treatment62Yoshiba T. Saga Y. Urabe M. Uchibori R. Matsubara S. Fujiwara H. Mizukami H. CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6.Oncol. 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