Precision Strikes on HIV: CRISPR/Cas9-Mediated Disruption of CCR5 and CXCR4 to Block Viral Entry and Establish Cellular Immunity
DOI:
https://doi.org/10.70749/ijbr.v3i6.1754Keywords:
HIV, Cellular Immunity, HIV Cure, CRISPR, Cas9Abstract
As of 2023, there were about 39 million people living with HIV, making it a persistent threat to global health. Lifelong treatment is required because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent reservoirs. By focusing on host co-receptors CCR5 and CXCR4, which are essential for HIV entry into CD4+ T cells and macrophages, the CRISPR/Cas9 gene-editing system provides a novel strategy. The disruption of CCR5 and CXCR4 by CRISPR/Cas9 to stop viral entry and build cellular immunity is thoroughly examined in this review. The design of guide RNAs, delivery methods, and off-target mitigation techniques are among the molecular mechanisms of CRISPR/Cas9 that we examine. While CXCR4 editing presents difficulties because of its wider physiological functions, preclinical research shows that CCR5 knockout in T cells and hematopoietic stem cells (HSCs) confers strong HIV resistance. Safe engraftment and lower viral loads are promising outcomes of clinical trials that target CCR5. We also talk about new technologies like base editing, scalability, viral escape risks, and ethical issues. This review identifies important barriers for clinical translation while highlighting the potential of CRISPR/Cas9 to provide a functional HIV cure.
Downloads
References
UNAIDS. (2023). Global HIV & AIDS statistics — 2023 fact sheet.
https://www.unaids.org/en/resources/fact-sheet
Deeks, S. G., Lewin, S. R., & Havlir, D. V. (2013). The end of AIDS: HIV infection as a chronic disease. The Lancet, 382(9903), 1525–1533.
https://doi.org/10.1016/S0140-6736(13)61809-7
Siliciano, J. D., & Siliciano, R. F. (2015). The latent reservoir for HIV-1 in resting CD4+ T cells and its implications for eradication strategies. Cell Host & Microbe, 18(6), 611–617.
https://doi.org/10.1016/j.chom.2015.11.007
Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096.
https://doi.org/10.1126/science.1258096
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., ... & Zhang, F. (2013). Multiplex genome engineering use CRISPR/Cas systems. Science, 339(6121), 819–823.
https://doi.org/10.1126/science.1231143
Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature Biotechnology, 34(9), 933–941.
https://doi.org/10.1038/nbt.3659
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.
https://doi.org/10.1126/science.1225829
Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., ... & Landau, N. R. (1996). Identification of a major co-receptor for primary isolates of HIV-1. Nature, 381(6584), 661–666.
https://doi.org/10.1038/381661a0
Hütter, G., Nowak, D., Mossner, M., Ganepola, S., Müßig, A., Allers, K., ... & Hofmann, J. (2009). Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. New England Journal of Medicine, 360(7), 692–698.
https://doi.org/10.1056/NEJMoa0802905
Feng, Y., Broder, C. C., Kennedy, P. E., & Berger, E. A. (1996). HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science, 272(5263), 872–877.
https://doi.org/10.1126/science.272.5263.872
Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262–1278.
https://doi.org/10.1016/j.cell.2014.05.010
Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), 347–355.
https://doi.org/10.1038/nbt.2842
Tebas, P., Stein, D., Tang, W. W., Frank, I., Wang, S. Q., Lee, G., ... & June, C. H. (2014). Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. New England Journal of Medicine, 370(10), 901–910.
https://doi.org/10.1056/NEJMoa1300662
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., ... & Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science, 339(6121), 823–826.
https://doi.org/10.1126/science.1232033
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636–646.
https://doi.org/10.1038/nrg2842
Yu, S., Yao, Y., Zhao, H., Wang, J., & Zhang, F. (2021). Multiplexed CRISPR/Cas9 editing of CCR5 and CXCR4 for HIV-1 resistance. Journal of Virology, 95(12), e02489-20.
https://doi.org/10.1128/JVI.02489-20
Tsai, S. Q., & Joung, J. K. (2016). Defining and improving the genome-wide specificity of CRISPR-Cas9 nucleases. Nature Reviews Genetics, 17(5), 300–312.
https://doi.org/10.1038/nrg.2016.28
Samson, M., Libert, F., Doranz, B. J., Rucker, J., Liesnard, C., Farber, C. M., ... & Parmentier, M. (1996). Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature, 382(6593), 722–725.
https://doi.org/10.1038/382722a0
Liu, R., Paxton, W. A., Choe, S., Ceradini, D., Martin, S. R., Horuk, R., ... & Landau, N. R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell, 86(3), 367–377.
https://doi.org/10.1016/S0092-8674(00)80110-5
Allers, K., Hütter, G., Hofmann, J., Loddenkemper, C., Rieger, K., Thiel, E., & Schneider, T. (2011). Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood, 117(10), 2791–2799.
https://doi.org/10.1182/blood-2010-09-309591
Ye, L., Wang, J., Beyer, A. I., Teque, F., Cradick, T. J., Qi, Z., ... & Kan, Y. W. (2014). Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation through genome editing. Stem Cells, 32(10), 2649–2659.
https://doi.org/10.1002/stem.1771
Xu, L., Yang, H., Gao, Y., Chen, Z., Xie, L., Li, Y., ... & Wang, J. (2017). CRISPR/Cas9-mediated CCR5 ablation in human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo. Molecular Therapy, 25(8), 1782–1789.
https://doi.org/10.1016/j.ymthe.2017.04.027
Kang, H., Minder, P., Park, M. A., Mesquitta, W. T., Torbett, B. E., & Slukvin, I. I. (2015). CCR5 disruption in induced pluripotent stem cells using CRISPR/Cas9 provides selective resistance of immune cells to CCR5-tropic HIV-1 virus. Molecular Therapy—Nucleic Acids, 4, e268.
https://doi.org/10.1038/mtna.2015.42
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.
https://doi.org/10.1038/nbt.2623
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., ... & Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology, 34(2), 184–191.
https://doi.org/10.1038/nbt.3437
Bleul, C. C., Wu, L., Hoxie, J. A., Springer, T. A., & Mackay, C. R. (1997). The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proceedings of the National Academy of Sciences, 94(5), 1925–1930.
https://doi.org/10.1073/pnas.94.5.1925
Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I., & Littman, D. R. (1998). Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature, 393(6685), 595–599.
Zhang, Y., Zhang, X., Cheng, C., Mu, W., Liu, X., Li, N., ... & Wei, W. (2017). CRISPR-Cas9-mediated CXCR4 silencing for HIV-1 therapy. Molecular Therapy—Nucleic Acids, 9, 65–74.
https://doi.org/10.1016/j.omtn.2017.09.004
Hou, P., Chen, S., Wang, S., Yu, X., Chen, Y., Li, M., ... & Liu, M. (2020). Genome editing of CXCR4 by CRISPR/Cas9 confers cells resistant to HIV-1 infection. Scientific Reports, 10(1), 16835.
https://doi.org/10.1038/s41598-020-73745-0
Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S. I., Kitamura, Y., ... & Tani, K. (1996). Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature, 382(6592), 635–638.
https://doi.org/10.1038/382635a0
Gao, Y., Yu, S., Liu, Z., Zhang, F., & Wang, J. (2020). CXCR4 knockout in hematopoietic stem cells impairs engraftment and immune reconstitution. Blood Advances, 4(15), 3612–3623.
https://doi.org/10.1182/bloodadvances.2020001789
Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., ... & Porteus, M. H. (2015). Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature Biotechnology, 33(9), 985–989.
https://doi.org/10.1038/nbt.3290
Liu, Z., Chen, S., Jin, X., Wang, Q., Yang, K., Li, C., ... & Wang, J. (2021). Dual targeting of CCR5 and CXCR4 by CRISPR/Cas9 confers resistance to both R5- and X4-tropic HIV-1. Molecular Therapy, 29(3), 1187–1199.
https://doi.org/10.1016/j.ymthe.2020.11.015
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587), 490–495.
https://doi.org/10.1038/nature16526
Naldini, L. (2015). Gene therapy returns to centre stage. Nature, 526(7573), 351–360.
https://doi.org/10.1038/nature15818
Wang, W., Ye, C., Liu, J., Zhang, D., Kim, J. T., & Wang, J. (2018). CCR5 gene disruption via lentiviral vectors expressing Cas9 and single-guide RNA renders cells resistant to HIV-1 infection. Molecular Therapy—Nucleic Acids, 13, 246–254.
https://doi.org/10.1016/j.omtn.2018.09.008
Yin, H., Kauffman, K. J., & Anderson, D. G. (2017). Delivery technologies for genome editing. Nature Reviews Drug Discovery, 16(6), 387–399.
https://doi.org/10.1038/nrd.2016.280
Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., ... & Marson, A. (2015). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences, 112(33), 10437–10442.
https://doi.org/10.1073/pnas.1512503112
Zhang, P., Narendran, S., Pereira, M., & Quan, D. (2020). Lipid nanoparticles for in vivo delivery of CRISPR/Cas9 components. Advanced Drug Delivery Reviews, 168, 135–148.
https://doi.org/10.1016/j.addr.2020.09.005
Wei, T., Cheng, Q., Min, Y. L., Olson, E. N., & Siegwart, D. J. (2020). Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue-specific genome editing. Nature Communications, 11(1), 3232.
https://doi.org/10.1038/s41467-020-17029-3
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4), 910–918.
https://doi.org/10.1016/j.cell.2013.04.025
Hultquist, J. F., Schumann, K., Woo, J. M., Manganaro, L., McGregor, M. J., Doudna, J. A., ... & Marson, A. (2016). CRISPR/Cas9 knockout of CCR5 protects against HIV-1 infection in humanized mice. Cell Reports, 17(9), 2169–2178.
https://doi.org/10.1016/j.celrep.2016.10.067
Li, C., Guan, X., Du, T., Jin, W., Wu, B., Liu, Y., ... & Wang, J. (2019). Inhibition of HIV-1 infection of primary CD4+ T-cells by CRISPR/Cas9-mediated CCR5 knockout. Frontiers in Immunology, 10, 1936.
https://doi.org/10.3389/fimmu.2019.01936
Holt, N., Wang, J., Kim, K., Friedman, G., Wang, X., Taupin, V., ... & Gregory, P. D. (2010). Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nature Biotechnology, 28(8), 839–847.
https://doi.org/10.1038/nbt.1663
Didigu, C. A., Wilen, C. B., Wang, J., Du, J., Secreto, A. J., Doms, R. W., ... & Young, J. A. T. (2014). Simultaneous zinc-finger nuclease editing of the HIV coreceptors CCR5 and CXCR4 protects CD4+ T cells from HIV-1 infection. Blood, 123(1), 61–69.
https://doi.org/10.1182/blood-2013-08-521377
Mandal, P. K., Ferreira, L. M., Collins, R., Meissner, T. B., Boutwell, C. L., Friesen, M., ... & Cowan, C. A. (2014). Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell, 15(5), 643–652.
https://doi.org/10.1016/j.stem.2014.10.004
Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., & Yang, S. H. (2015). Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy—Nucleic Acids, 4, e264.
https://doi.org/10.1038/mtna.2015.37
Liu, Z., Chen, S., Jin, X., Wang, Q., Yang, K., Li, C., ... & Wang, J. (2021). Dual targeting of CCR5 and CXCR4 by CRISPR/Cas9 confers resistance to both R5- and X4-tropic HIV-1. Molecular Therapy, 29(3), 1187–1199.
https://doi.org/10.1016/j.ymthe.2020.11.015
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268), 84–88.
https://doi.org/10.1126/science.aad5227
Peterson, C. W., Wang, J., Norman, K. K., Norgaard, Z. K., Humbert, O., Tse, C. K., ... & Kiem, H. P. (2016). Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates. Blood, 127(20), 2416–2426.
https://doi.org/10.1182/blood-2015-09-672337
Kiem, H. P., Jerome, K. R., Deeks, S. G., & McCune, J. M. (2012). Hematopoietic-stem-cell-based gene therapy for HIV disease. Cell Stem Cell, 10(2), 137–147.
https://doi.org/10.1016/j.stem.2011.12.012
Wang, J., Exline, C. M., DeClercq, J. J., Llewellyn, G. N., Hayward, S. B., Li, P. W. L., ... & Cannon, P. M. (2015). Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nature Biotechnology, 33(12), 1256–1263.
https://doi.org/10.1038/nbt.3408
Xu, L., Wang, J., Liu, Y., Xie, L., Su, B., Mou, D., ... & Yang, H. (2019). CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. New England Journal of Medicine, 381(13), 1240–1247.
https://doi.org/10.1056/NEJMoa1817426
Tebas, P., Jadlowsky, J. K., Shaw, P. A., Tian, L., Esparza, E., Brennan, A. L., ... & June, C. H. (2021). CCR5-edited CD4+ T cells for HIV therapy: A phase I clinical trial. Science Translational Medicine, 13(605), eaba8689.
https://doi.org/10.1126/scitranslmed.aba8689
Gupta, R. K., Abdul-Jawad, S., McCoy, L. E., Mok, H. P., Peppa, D., Salgado, M., ... & Frater, J. (2019). HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation. Nature, 568(7751), 244–248.
https://doi.org/10.1038/s41586-019-1027-4
Hütter, G., & Thiel, E. (2011). Allogeneic transplantation of CCR5-deficient progenitor cells in a patient with HIV infection: An update. AIDS, 25(6), 825–826.
https://doi.org/10.1097/QAD.0b013e3283448859
Cornu, T. I., Mussolino, C., & Cathomen, T. (2017). Refining strategies to translate genome editing to the clinic. Nature Medicine, 23(4), 415–423.
https://doi.org/10.1038/nm.4313
Wang, Q., Chen, S., Xiao, Q., Liu, Z., Liu, S., Wang, J., ... & Yang, H. (2022). CXCR4-edited T cells in HIV therapy: Preliminary safety and efficacy data. Journal of Infectious Diseases, 226(4), 614–623.
https://doi.org/10.1093/infdis/jiaa512
Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacoby, K., Collingwood, M. A., ... & Porteus, M. H. (2018). A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem cells. Nature Medicine, 24(8), 1216–1224.
https://doi.org/10.1038/s41591-018-0137-0
Margolis, D. M., Garcia, J. V., Hazuda, D. J., & Haynes, B. F. (2016). Latency reversal and viral clearance to cure HIV-1. Science, 353(6297), aaf6517.
https://doi.org/10.1126/science.aaf6517
Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., ... & Porteus, M. H. (2019). Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature Medicine, 25(2), 249–254.
https://doi.org/10.1038/s41591-018-0326-x
Cyranoski, D. (2016). CRISPR gene-editing tested in a person for the first time. Nature, 539(7630), 479.
https://doi.org/10.1038/nature.2016.20988
Wagner, D. L., Amini, L., Wendering, D. J., Burkhardt, L. M., Akyüz, L., Reinke, P., ... & Schmueck-Henneresse, M. (2019). High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nature Medicine, 25(2), 242–248.
https://doi.org/10.1038/s41591-018-0204-6
Baylis, F., & McLeod, M. (2017). First-in-human Phase 1 CRISPR gene editing cancer trials: Are we ready? Current Gene Therapy, 17(4), 309–319.
https://doi.org/10.2174/1566523217666171121165935
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.
https://doi.org/10.1126/science.aab1028
Lanphier, E., Urnov, F., Haecker, S. E., Werner, M., & Smolenski, J. (2015). Don’t edit the human germ line. Nature, 519(7544), 410–411.
https://doi.org/10.1038/519410a
Jasanoff, S., & Hurlbut, J. B. (2018). A global observatory for gene editing. Nature, 555(7697), 435–437.
https://doi.org/10.1038/d41586-018-03270-w
Dzau, V. J., McCabe, C., & Balatbat, C. (2020). Ensuring equitable access to gene therapies. Nature Medicine, 26(12), 1829–1832.
https://doi.org/10.1038/s41591-020-01148-z
Moon, S., & Khushf, G. (2016). Global health equity and the role of gene editing. The Lancet Global Health, 4(12), e845–e846.
https://doi.org/10.1016/S2214-109X(16)30234-9
World Health Organization. (2021). Human genome editing: Recommendations. Retrieved from
https://www.who.int/publications/i/item/9789240030381
Arts, E. J., & Hazuda, D. J. (2012). HIV-1 antiretroviral drug resistance: Mechanisms and clinical implications. Nature Reviews Microbiology, 10(8), 558–566.
https://doi.org/10.1038/nrmicro2837
Moore, J. P., & Klasse, P. J. (2008). HIV-1 coreceptor use: The interplay of viral and host factors. Cell Host & Microbe, 4(6), 509–511.
https://doi.org/10.1016/j.chom.2008.11.002
Kaminski, R., Chen, Y., Fischer, T., Tedaldi, E., Napoli, A., Zhang, Y., ... & Khalili, K. (2016). Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing. Scientific Reports, 6, 22555.
https://doi.org/10.1038/srep22555
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420–424.
https://doi.org/10.1038/nature17946
Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., ... & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157.
https://doi.org/10.1038/s41586-019-1711-4
Zhang, P., Narendran, S., Pereira, M., & Quan, D. (2020). Lipid nanoparticles for in vivo delivery of CRISPR/Cas9 components. Advanced Drug Delivery Reviews, 168, 135–148.
https://doi.org/10.1016/j.addr.2020.09.005
Margolis, D. M., Garcia, J. V., Hazuda, D. J., & Haynes, B. F. (2016). Latency reversal and viral clearance to cure HIV-1. Science, 353(6297), aaf6517.
https://doi.org/10.1126/science.aaf6517
Peterson, C. W., Wang, J., Norman, K. K., Norgaard, Z. K., Humbert, O., Tse, C. K., ... & Kiem, H. P. (2016). Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates. Blood, 127(20), 2416–2426. https://doi.org/10.1182/blood-2015-09-672337
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H. W., Listgarten, J., & Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology, 34(2), 184–191.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Indus Journal of Bioscience Research

This work is licensed under a Creative Commons Attribution 4.0 International License.
