Clustered Regularly Interspaced Short Palindromic Repeat Paired Associated Protein 9 (CRISPR-Cas9) System and Its Opportunity in Medical Science - A Narrative Review
Article Sidebar
Main Article Content
Abstract
The clustered regularly interspaced short palindromic repeat paired associated protein 9 (CRISPR-Cas9) is a site-specific genome editing tool that enables scientists to edit or introduce genetic mutation at will. CRISPR-Cas9 consists of two essential key players; a programmable RNA called single guide RNA (sgRNA) and the Cas9 protein which functions as a molecular scissors that does the cutting. Since its discovery, CRISPR-Cas9 has received vast attention due to its simplicity, convenience, and superior precision of use. Its application extends into various fields including the health sciences where it has been used to enhance the understanding of pathogenesis and help in therapeutic intervention. Despite the promising potentials and applications of CRISPR-Cas9, there are several aspects that need to be addressed including the method of delivery, off-target cutting and ethical issues in human germline modification. The purposes of this review are to perform a comprehensive literature search of publications on the CRISPR-Cas9 system and to highlight potential applications of CRISPR-Cas9 in the field of medical sciences. In this present review, we discuss the background of CRISPR-Cas9, its mechanisms of genome modification and its applications in the medical field including its use in the study of animal model production, genetics, multifactorial and complex diseases. In addition, we also discuss the limitations associated with CRISPR-Cas9 application. CRISPR-Cas9 has accelerated medical studies and facillitate the collection of vast amounts of information. However, its limitations should be further studied in order to reap its greatest benefits.
Keywords: CRISPR-Cas9; CRISPR-Cas9 applications; genome editing technique; site-specific genome editing tool; therapeutic intervention
*Corresponding author: Tel.: (+06) 03 32584743 Fax: (+06) 03 3258 4602
E-mail: [email protected]
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Copyright Transfer Statement
The copyright of this article is transferred to Current Applied Science and Technology journal with effect if and when the article is accepted for publication. The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, electronic form (offline, online) or any other reproductions of similar nature.
The author warrants that this contribution is original and that he/she has full power to make this grant. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors.
Here is the link for download: Copyright transfer form.pdf
References
Zarei, A., Razban, V., Hosseini, S.E. and Tabei, S., 2019. Creating cell and animal models of human disease by genome editing using CRISPR/Cas9. The Journal of Gene Medicine, 21(4), e3082, https://doi.org/10.1002/jgm.3082.
Peng, R., Lin, G. and Li, J., 2016. Potential pitfalls of CRISPR/Cas9-mediated genome editing. The FEBS Journal, 283(7), 1218-1231.
Zhao, J., Fang, H. and Zhang, D., 2020. Expanding application of CRISPR-Cas9 system in microorganisms. Synthetic and Systems Biotechnology, 5(4), 269-276.
Gupta, D., Bhattacharjee, O., Mandal, D., Sen, M.K., Dey, D., Dasgupta, A., Kazi, T.A., Gupta, R., Sinharoy, S., Acharya, K., Chattopadhyay, D., Ravichandiran, V., Roy, S. and Ghosh, D., 2019. CRISPR-Cas9 system: A new-fangled dawn in gene editing. Life Sciences, 232, 116636, https://doi.org/10.1016/j.lfs.2019.116636.
Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F. and Jaenisch, R., 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4), 910-918.
Cho, B., Kim, S.J., Lee, E.J., Ahn, S.M., Lee, J.S., Ji, D.Y., Lee, K. and Kang, J.T., 2018. Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system. Transgenic Research, 27(3), 289-300.
Kotagama, O.W., Jayasinghe, C.D. and Abeysinghe, T., 2019. Era of genomic medicine: A narrative review on CRISPR technology as a potential therapeutic tool for human diseases. BioMed Research International, 2019, 1369682, https://doi.org/10.1155/2019/1369682.
Firth, A.L., Menon, T., Parker, G.S., Qualls, S.J., Lewis, B.M., Ke, E., Dargitz, C.T., Wright, R., Khanna, A., Gage, F.H. and Verma, I.M., 2015. Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient iPSCs. Cell Reports, 12(9), 1385-1390.
Niu, X., He, W., Song, B., Ou, Z., Fan, D., Chen, Y., Fan, Y. and Sun, X., 2016. Combining single strand oligodeoxynucleotides and CRISPR/Cas9 to correct gene mutations in β-thalassemia-induced pluripotent stem cells. The Journal of Biological Chemistry, 291(32), 16576-16585.
Song, B., Fan, Y., He, W., Zhu, D., Niu, X., Wang, D., Ou, Z., Luo, M. and Sun, X., 2015. Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells and Development, 24(9), 1053-1065.
Pavani, G., Fabiano, A., Laurent, M., Amor, F., Cantelli, E., Chalumeau, A., Maule, G., Tachtsidi, A., Concordet, J.P., Cereseto, A., Mavilio, F., Ferrari, G., Miccio, A. and Amendola, M., 2021. Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells. Blood Advances, 5(5), 1137-1153.
Hu, Z., Yu, L., Zhu, D., Ding, W., Wang, X., Zhang, C., Wang, L., Jiang, X., Shen, H., He, D., Li, K., Xi, L., Ma, D. and Wang, H., 2014. Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. BioMed Research International, 2014, 612823, https://doi.org/10.1155/2014/612823.
Chen, Y. and Zhang, Y., 2018. Application of the CRISPR/Cas9 system to drug resistance in breast cancer. Advanced Science, 5(6), 1700964, https://doi.org/10.1002/advs.201700964.
Hannafon, B.N., Cai, A., Calloway, C.L., Xu, Y.F., Zhang, R., Fung, K.M. and Ding, W.Q., 2019. miR-23b and miR-27b are oncogenic microRNAs in breast cancer: evidence from a CRISPR/Cas9 deletion study. BMC Cancer, 19(1), 642, https://doi.org/10.1186/s12885-019-5839-2.
Liu, C., Zhang, L., Liu, H. and Cheng, K., 2017. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. Journal of Controlled Release, 266, 17-26.
Porteus M. H., 2015. Towards a new era in medicine: therapeutic genome editing. Genome Biology, 16, 286, https://doi.org/10.1186/s13059-015-0859-y.
Cai, L., Fisher, A.L., Huang, H. and Xie, Z., 2016. CRISPR-mediated genome editing and human diseases. Genes and Diseases, 3(4), 244-251.
Ishino, Y., Krupovic, M. and Forterre, P., 2018. History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. Journal of Bacteriology, 200(7), e00580-17, https://doi.org/10.1128/JB.00580-17.
Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. and 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.
Makarova, K.S., Wolf, Y.I., Iranzo, J., Shmakov, S.A., Alkhnbashi, O.S., Brouns, S., Charpentier, E., Cheng, D., Haft, D.H., Horvath, P., Moineau, S., Mojica, F., Scott, D., Shah, S.A., Siksnys, V., Terns, M.P., Venclovas, Č., White, M.F., Yakunin, A.F., Yan, W. and Koonin, E.V., 2020. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67-83.
Tang, Y. and Fu, Y., 2018. Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing. Cell and Bioscience, 8, 59, https://doi.org/10.1186/s13578-018-0255-x.
Jiang, F. and Doudna, J.A., 2017. CRISPR-Cas9 structures and mechanisms. Annual Review of Biophysics, 46, 505-529.
Cao, J., Xiao, Q. and Yan, Q., 2018. The multiplexed CRISPR targeting platforms. Drug Discovery Today: Technologies, 28, 53-61.
Porteus, M., 2016. Genome editing: A new approach to human therapeutics. Annual Review of Pharmacology and Toxicology, 56, 163-190.
Cubbon, A., Ivancic-Bace, I. and Bolt, E.L., 2018. CRISPR-Cas immunity, DNA repair and genome stability. Bioscience Reports, 38(5), https://doi.org/10.1042/BSR20180457.
Rodriguez, E., 2016. Ethical issues in genome editing using Crispr/Cas9 system. Journal of Clinical Research and Bioethics, 7(2), https://doi.org/10.4172/2155-9627.1000266.
Shen, B., Zhang, J., Wu, H., Wang, J., Ma, K., Li, Z., Zhang, X., Zhang, P. and Huang, X., 2013. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 23(5), 720-723.
Shao, Y., Guan, Y., Wang, L., Qiu, Z., Liu, M., Chen, Y., Wu, L., Li, Y., Ma, X., Liu, M. and Li, D., 2014. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nature Protocols, 9(10), 2493-2512.
Niu, Y., Shen, B., Cui, Y., Chen, Y., Wang, J., Wang, L., Kang, Y., Zhao, X., Si, W., Li, W., Xiang, A.P., Zhou, J., Guo, X., Bi, Y., Si, C., Hu, B., Dong, G., Wang, H., Zhou, Z., Li, T. and Sha, J., 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156(4), 836-843.
Fan, Z., Perisse, I.V., Cotton, C.U., Regouski, M., Meng, Q., Domb, C., Van Wettere, A.J., Wang, Z., Harris, A., White, K.L. and Polejaeva, I.A., 2018. A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene. JCI Insight, 3(19), e123529, https://doi.org/10.1172/jci.insight.123529.
Kim, H., Kim, M., Im, S.K. and Fang, S., 2018. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Laboratory Animal Research, 34(4), 147-159.
Huang, J., Chen, M., Whitley, M.J., Kuo, H.C., Xu, E.S., Walens, A., Mowery, Y.M., Van Mater, D., Eward, W.C., Cardona, D.M., Luo, L., Ma, Y., Lopez, O.M., Nelson, C.E., Robinson-Hamm, J.N., Reddy, A., Dave, S.S., Gersbach, C.A., Dodd, R.D. and Kirsch, D.G., 2017. Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma. Nature Communications, 8, 15999, https://doi.org/10.1038/ncomms15999.
Xue, W., Chen, S., Yin, H., Tammela, T., Papagiannakopoulos, T., Joshi, N.S., Cai, W., Yang, G., Bronson, R., Crowley, D.G., Zhang, F., Anderson, D.G., Sharp, P.A. and Jacks, T., 2014. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature, 514(7522), 380-384.
Cooney, A.L., McCray, P.B. Jr. and Sinn, P.L., 2018. Cystic fibrosis gene therapy: Looking back, looking forward. Genes, 9(11), 538, https://doi.org/10.3390/genes9110538.
Mall, M.A. and Hartl, D., 2014. CFTR: cystic fibrosis and beyond. The European Respiratory Journal, 44(4), 1042-1054.
Odera, M., Furuta, T., Sohma, Y. and Sakurai, M., 2018. Molecular dynamics simulation study on the structural instability of the most common cystic fibrosis-associated mutant ΔF508-CFTR. Biophysics and Physicobiology, 15, 33-44.
Sondo, E., Falchi, F., Caci, E., Ferrera, L., Giacomini, E., Pesce, E., Tomati, V., Mandrup Bertozzi, S., Goldoni, L., Armirotti, A., Ravazzolo, R., Cavalli, A. and Pedemonte, N., 2018. Pharmacological inhibition of the ubiquitin ligase RNF5 rescues F508del-CFTR in cystic fibrosis airway epithelia. Cell Chemical Biology, 25(7), 891-905.
Schwank, G., Koo, B.K., Sasselli, V., Dekkers, J.F., Heo, I., Demircan, T., Sasaki, N., Boymans, S., Cuppen, E., van der Ent, C.K., Nieuwenhuis, E.E., Beekman, J.M. and Clevers, H., 2013. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 13(6), 653-658.
Smirnikhina, S.A., Kondrateva, E.V., Adilgereeva, E.P., Anuchina, A.A., Zaynitdinova, M.I., Slesarenko, Y.S., Ershova, A.S., Ustinov, K.D., Yasinovsky, M.I., Amelina, E.L., Voronina, E.S., Yakushina, V.D., Tabakov, V.Y. and Lavrov, A.V., 2020. P.F508del editing in cells from cystic fibrosis patients. PloS One, 15(11), e0242094, https://doi.org/10.1371/journal.pone.0242094.
Wattanapanitch M., 2021. Correction of hemoglobin E/beta-thalassemia patient-derived iPSCs using CRISPR/Cas9. Methods in Molecular Biology, 2211, 193-211.
Wattanapanitch, M., Damkham, N., Potirat, P., Trakarnsanga, K., Janan, M., U-Pratya, Y., Kheolamai, P., Klincumhom, N. and Issaragrisil, S., 2018. One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system. Stem Cell Research & Therapy, 9(1), 46, https://doi.org/10.1186/s13287-018-0779-3.
Ali, G., Tariq, M.A., Shahid, K., Ahmad, F.J. and Akram, J., 2021. Advances in genome editing: the technology of choice for precise and efficient β-thalassemia treatment. Gene Therapy, 28(1-2), 6-15.
Srivastava, A. and Shaji, R.V., 2017. Cure for thalassemia major - from allogeneic hematopoietic stem cell transplantation to gene therapy. Haematologica, 102(2), 214-223.
Frangoul, H., Altshuler, D., Cappellini, M.D., Chen, Y.S., Domm, J., Eustace, B.K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., Ho, T.W., Kattamis, A., Kernytsky, A., Lekstrom-Himes, J., Li, A.M., Locatelli, F., Mapara, M.Y., de Montalembert, M., Rondelli, D., Sharma, A. and Corbacioglu, S., 2021. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. The New England Journal of Medicine, 384(3), 252-260.
Fialkowski, A., Beasley, T.M. and Tiwari, H.K., 2019. Multifactorial inheritance and complex diseases. In: R.E. Pyeritz, B.R. Korf and W.W. Grody, eds. Emery and Rimoin's Principles and Practice of Medical Genetics and Genomics: Foundations. 7th ed. London: Academic Press, pp. 323-358.
Chen, M., Mao, A., Xu, M., Weng, Q., Mao, J. and Ji, J., 2019. CRISPR-Cas9 for cancer therapy: Opportunities and challenges. Cancer Letters, 447, 48-55.
Zhen, S., Hua, L., Takahashi, Y., Narita, S., Liu, Y.H. and Li, Y., 2014. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochemical and Biophysical Research Communications, 450(4), 1422-1426.
Ahmed, M., Daoud, G.H., Mohamed, A. and Harati, R., 2021. New Insights into the therapeutic applications of CRISPR/Cas9 genome editing in breast cancer. Genes, 12(5), 723, https://doi.org/10.3390/genes12050723.
An, Y., Zhang, Z., Shang, Y., Jiang, X., Dong, J., Yu, P., Nie, Y. and Zhao, Q., 2015. miR-23b-3p regulates the chemoresistance of gastric cancer cells by targeting ATG12 and HMGB2. Cell Death and Disease, 6(5), e1766, https://doi.org/10.1038/cddis.2015.123.
Qi, P., Xu, M.D., Shen, X.H., Ni, S.J., Huang, D., Tan, C., Weng, W.W., Sheng, W.Q., Zhou, X.Y. and Du, X., 2015. Reciprocal repression between TUSC7 and miR-23b in gastric cancer. International Journal of Cancer, 137(6), 1269-1278.
Chen, L., Li, H., Han, L., Zhang, K., Wang, G., Wang, Y., Liu, Y., Zheng, Y., Jiang, T., Pu, P., Jiang, C. and Kang, C., 2011. Expression and function of miR-27b in human glioma. Oncology Reports, 26(6), 1617-1621.
Fanning, S.W., Mayne, C.G., Dharmarajan, V., Carlson, K.E., Martin, T.A., Novick, S.J., Toy, W., Green, B., Panchamukhi, S., Katzenellenbogen, B.S., Tajkhorshid, E., Griffin, P. R., Shen, Y., Chandarlapaty, S., Katzenellenbogen, J.A. and Greene, G.L., 2016. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation. ELife, 5, e12792, https://doi.org/10.7554/eLife.12792.
Martinez-Lage, M., Torres-Ruiz, R., Puig-Serra, P., Moreno-Gaona, P., Martin, M.C., Moya, F.J., Quintana-Bustamante, O., Garcia-Silva, S., Carcaboso, A.M., Petazzi, P., Bueno, C., Mora, J., Peinado, H., Segovia, J.C., Menendez, P. and Rodriguez-Perales, S., 2020. In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nature Communications, 11(1), 5060, https://doi.org/10.1038/s41467-020-18875-x.
Xiao, Q., Guo, D. and Chen, S., 2019. Application of CRISPR/Cas9-based gene editing in HIV-1/AIDS therapy. Frontiers in Cellular and Infection Microbiology, 9, 69, https://doi.org/10.3389/fcimb.2019.00069.
Das, A.T., Binda, C.S. and Berkhout, B., 2019. Elimination of infectious HIV DNA by CRISPR-Cas9. Current Opinion in Virology, 38, 81-88.
Liang, C., Wainberg, M.A., Das, A.T. and Berkhout, B., 2016. CRISPR/Cas9: a double-edged sword when used to combat HIV infection. Retrovirology, 13(1), 37, https://doi.org/10.1186/s12977-016-0270-0.
Huang, S.H., Ren, Y., Thomas, A.S., Chan, D., Mueller, S., Ward, A.R., Patel, S., Bollard, C.M., Cruz, C.R., Karandish, S., Truong, R., Macedo, A.B., Bosque, A., Kovacs, C., Benko, E., Piechocka-Trocha, A., Wong, H., Jeng, E., Nixon, D.F., Ho, Y.C. and Jones, R.B., 2018. Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells. The Journal of Clinical Investigation, 128(2), 876-889.
Panfil, A.R., London, J.A., Green, P.L. and Yoder, K.E., 2018. CRISPR/Cas9 genome editing to disable the latent HIV-1 provirus. Frontiers in Microbiology, 9, 3107, https://doi.org/10.3389/fmicb.2018.03107.
Zhu, W., Lei, R., Le Duff, Y., Li, J., Guo, F., Wainberg, M.A. and Liang, C., 2015. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology, 12, 22, https://doi.org/10.1186/s12977-015-0150-z.
Ebina, H., Misawa, N., Kanemura, Y. and Koyanagi, Y., 2013. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Scientific Reports, 3, 2510, https://doi.org/10.1038/srep02510.
Yin, L., Hu, S., Mei, S., Sun, H., Xu, F., Li, J., Zhu, W., Liu, X., Zhao, F., Zhang, D., Cen, S., Liang, C. and Guo, F., 2018. CRISPR/Cas9 inhibits multiple steps of HIV-1 infection. Human Gene Therapy, 29(11), 1264-1276.
Wang, Z., Pan, Q., Gendron, P., Zhu, W., Guo, F., Cen, S., Wainberg, M.A. and Liang, C., 2016. CRISPR/Cas9-derived mutations both inhibit HIV-1 replication and accelerate viral escape. Cell Reports, 15(3), 481-489.
Yin, C., Zhang, T., Qu, X., Zhang, Y., Putatunda, R., Xiao, X., Li, F., Xiao, W., Zhao, H., Dai, S., Qin, X., Mo, X., Young, W.B., Khalili, K. and Hu, W., 2017. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Molecular Therapy, 25(5), 1168-1186.
Lino, C.A., Harper, J.C., Carney, J.P. and Timlin, J.A., 2018. Delivering CRISPR: a review of the challenges and approaches. Drug Delivery, 25(1), 1234-1257.
Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A. and Liu, D.R., 2013. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology, 31(9), 839-843.
Bessodes, M., Dhotel, H. and Mignet, N., 2019. Lipids for nucleic acid delivery: Cationic or neutral lipoplexes, synthesis, and particle formation. Methods in Molecular Biology, 1943, 123-139.
Nelson, C.E. and Gersbach, C.A., 2016. Engineering delivery vehicles for genome editing. Annual Review of Chemical and Biomolecular Engineering, 7, 637-662.
Moss, K.H., Popova, P., Hadrup, S.R., Astakhova, K. and Taskova, M., 2019. Lipid nanoparticles for delivery of therapeutic RNA oligonucleotides. Molecular Pharmaceutics 16(6), 2265-2277.
Cheng, Q., Wei, T., Farbiak, L., Johnson, L.T., Dilliard, S.A. and Siegwart, D.J., 2020. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Natural Nanotechnology, 15(4), 313-20.
Behr, M., Zhou, J., Xu, B. and Zhang, H., 2021. In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges. Acta Pharmaceutica Sinica B, 11(8), 2150-2171.
Modrzejewski, D., Hartung, F., Lehnert, H., Sprink, T., Kohl, C., Keilwagen, J. and Wilhelm, R., 2020. Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas: A systematic review in plants. Frontiers in Plant Science, 11, 574959, https://doi.org/10.3389/fpls.2020.574959.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A. and Zhang, F., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823.
Jiang, W., Bikard, D., Cox, D., Zhang, F. and Marraffini, L.A., 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233-239.
Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K. and Sander, J.D., 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31(9), 822-826.
Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao, G. and Zhang, F., 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 31(9), 827-832.
Tang, L., Zeng, Y., Du, H., Gong, M., Peng, J., Zhang, B., Lei, M., Zhao, F., Wang, W., Li, X. and Liu, J., 2017. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Molecular Genetics and Genomics, 292(3), 525-533.
Aryal, N.K., Wasylishen, A.R. and Lozano, G., 2018. CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo. Cell Death and Disease, 9(11), 1099, https://doi.org/10.1038/s41419-018-1146-0.
Naeem, M., Majeed, S., Hoque, M.Z. and Ahmad, I., 2020. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells, 9(7), 1608, httos://doi.org/10.3390/cells9071608.
Brokowski, C. and Adli, M., 2019. CRISPR ethics: Moral considerations for applications of a powerful tool. Journal of Molecular Biology, 431(1), 88-101.
Vassena, R., Heindryckx, B., Peco, R., Pennings, G., Raya, A., Sermon, K. and Veiga, A., 2016. Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells. Human Reproduction Update, 22(4), 411-419.
Billings, P.R., Hubbard, R. and Newman, S.A., 1999. Human germline gene modification: a dissent. Lancet, 353(9167), 1873-1875.