Abstract
CRISPR–Cas9 genome editing technology is a promising tool for genetically engineering immune cells and modulating immune systems. Although ex vivo genome editing of immune cells has reached clinical trials, in vivo application is still restricted by the instability and inefficient delivery of CRISPR–Cas9 components to immune cells through circulation. In this Review, we summarize ex vivo and in vivo strategies to deliver CRISPR–Cas9 components to both non-immune and immune cells. We review the progress made in non-immune cells because it offers insights that can be applied to advancing research in immune cells. We also discuss principles and challenges of immune system modulation using CRISPR–Cas9 genome editing technology.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
27,99 € / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
118,99 € per year
only 9,92 € per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Jacobson, D. L., Gange, S. J., Rose, N. R. & Graham, N. M. Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clin. Immunol. Immunopathol. 84, 223–243 (1997).
Marrack, P., Kappler, J. & Kotzin, B. L. Autoimmune disease: why and where it occurs. Nat. Med. 7, 899–905 (2001).
Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).
Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843–1846 (2011).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Guo, C., Ma, X., Gao, F. & Guo, Y. Off-target effects in CRISPR/Cas9 gene editing. Front. Bioeng. Biotechnol. 11, 1143157 (2023).
Kouranova, E. et al. CRISPRs for optimal targeting: delivery of CRISPR components as DNA, RNA, and protein into cultured cells and single-cell embryos. Hum. Gene Ther. 27, 464–475 (2016).
Kumar, R. et al. Polymeric delivery of therapeutic nucleic acids. Chem. Rev. 121, 11527–11652 (2021).
Sahel, D. K. et al. CRISPR/Cas9 genome editing for tissue-specific in vivo targeting: nanomaterials and translational perspective. Adv. Sci. 10, e2305072 (2023).
Pesch, T. et al. Molecular design, optimization, and genomic integration of chimeric B cell receptors in murine B cells. Front. Immunol. 10, 2630 (2019).
Yang, Y. A.-O., Wang, D., Lü, P., Ma, S. & Chen, K. Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system. Mol. Biol. Rep. 50, 3723–3738 (2019).
Senthilnathan, R. A.-O. X. et al. An update on CRISPR-Cas12 as a versatile tool in genome editing. Mol. Biol. Rep. 50, 2865–2881 (2019).
Liu, H., Zhu, Y., Li, M. & Gu, Z. A.-O. Precise genome editing with base editors. Med. Rev. 22, 75–84 (2019).
Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J. & Liu, D. R. Designing and executing prime editing experiments in mammalian cells. Nat. Protoc. 17, 2431–2468 (2022).
Zhao, Z., Shang, P., Mohanraju, P. & Geijsen, N. Prime editing: advances and therapeutic applications. Trends Biotechnol. 41, 1000–1012 (2022).
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023).
Zhou, T. et al. Lupus enhancer risk variant causes dysregulation of IRF8 through cooperative lncRNA and DNA methylation machinery. Nat. Commun. 13, 1855 (2022).
Bhowmik, R. & Chaubey, B. CRISPR/Cas9: a tool to eradicate HIV-1. AIDS Res. Ther. 19, 58 (2022).
Stefanoudakis, D. et al. The potential revolution of cancer treatment with CRISPR technology. Cancers 15, 1813 (2023).
Legut, M., Dolton, G., Mian, A. A., Ottmann, O. G. & Sewell, A. K. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood 131, 311–322 (2018).
Ray, M. et al. CRISPRed macrophages for cell-based cancer immunotherapy. Bioconjug. Chem. 29, 445–450 (2018).
Xu, C. et al. Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases. Nat. Commun. 9, 4092 (2018).
Limanskiy, V., Vyas, A., Chaturvedi, L. S. & Vyas, D. Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease. World J. Gastroenterol. 25, 2177–2187 (2019).
Bevacqua, R. J. et al. CRISPR-based genome editing in primary human pancreatic islet cells. Nat. Commun. 12, 2397 (2021).
Baker, C. & Hayden, M. S. Gene editing in dermatology: harnessing CRISPR for the treatment of cutaneous disease. F1000Res. 9, 281 (2020).
Lin, W. et al. Tumor-intrinsic YTHDF1 drives immune evasion and resistance to immune checkpoint inhibitors via promoting MHC-I degradation. Nat. Commun. 14, 265 (2023).
Levy, E. C., J., Reger, R., Allan, D. & Childs, R. RNA-seq analysis reveals CCR5 as a key target for CRISPR gene editing to regulate in vivo NK cell trafficking. Cancers 13, 872 (2021).
Guo, X. et al. CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy. J. Immunother. Cancer 9, e001975 (2021).
Greiner, V. et al. CRISPR-mediated editing of the B cell receptor in primary human B cells. iScience 12, 369–378 (2019).
Zhang, H. et al. CRISPR/Cas9-mediated gene editing in human iPSC-derived macrophage reveals lysosomal acid lipase function in human macrophages — brief report. Arterioscler. Thromb. Vasc. Biol. 37, 2156–2160 (2017).
Parnas, O. et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015).
Dimitri, A., Herbst, F. & Fraietta, J. A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol. Cancer 21, 78 (2022).
Nussing, S. et al. Efficient CRISPR/Cas9 gene editing in uncultured naive mouse T cells for in vivo studies. J. Immunol. 204, 2308–2315 (2020).
Zhang, J. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature 609, 369–374 (2022).
Ghaffari, S., Khalili, N. & Rezaei, N. CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy. J. Exp. Clin. Cancer Res. 40, 269 (2021).
Hu, Y. et al. Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory non-Hodgkin’s lymphoma: a first-in-human phase I study. EClinicalMedicine 60, 102010 (2023).
Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).
Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).
Lefesvre, P., Attema, J. & van Bekkum, D. A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer. BMC Mol. Biol. 3, 12 (2002).
Netea, M. G. et al. Defining trained immunity and its role in health and disease. Nat. Rev. Immunol. 20, 375–388 (2020).
Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019).
Blanco, E., Shen, H. & Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951 (2015).
Schudel, A., Francis, D. M. & Thomas, S. N. Material design for lymph node drug delivery. Nat. Rev. Mater. 4, 415–428 (2019).
Bulcaen, M. & Carlon, M. S. Gene editing flows to the lungs. Science 384, 1175–1176 (2019).
Lieleg, O., Baumgartel, R. M. & Bausch, A. R. Selective filtering of particles by the extracellular matrix: an electrostatic bandpass. Biophys. J. 97, 1569–1577 (2009).
Lechardeur, D. & Lukacs, G. L. Intracellular barriers to non-viral gene transfer. Curr. Gene Ther. 2, 183–194 (2002).
Shui, S., Wang, S. & Liu, J. Systematic investigation of the effects of multiple SV40 nuclear localization signal fusion on the genome editing activity of purified SpCas9. Bioengineering 9, 83 (2022).
Alberts B. et al. in Molecular Biology of the Cell Ch. 2, Ch. 12 (Garland Science, 2002).
Lin, Y., Wagner, E. & Lachelt, U. Non-viral delivery of the CRISPR/Cas system: DNA versus RNA versus RNP. Biomater. Sci. 10, 1166–1192 (2022).
Liu, C., Zhang, L., Liu, H. & Cheng, K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J. Control. Release 266, 17–26 (2017).
Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L. & Gao, G. Viral vector platforms within the gene therapy landscape. Signal Transduct. Target. Ther. 6, 53 (2021).
Fajrial, A. K., He, Q. Q., Wirusanti, N. I., Slansky, J. E. & Ding, X. A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing. Theranostics 10, 5532–5549 (2020).
Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, 252–260 (2021).
Philippidis, A. CASGEVY makes history as FDA approves first CRISPR/Cas9 genome edited therapy. Hum. Gene Ther. 35, 1–4 (2024).
Zhang, P., Zhang, G. & Wan, X. Challenges and new technologies in adoptive cell therapy. J. Hematol. Oncol. 16, 97 (2023).
Hamilton, A. G., Swingle, K. L. & Mitchell, M. J. Biotechnology: overcoming biological barriers to nucleic acid delivery using lipid nanoparticles. PLoS Biol. 21, e3002105 (2023).
Asmamaw Mengstie, M. Viral vectors for the in vivo delivery of CRISPR components: advances and challenges. Front. Bioeng. Biotechnol. 10, 895713 (2022).
Yu, J. et al. Design of a self-driven probiotic-CRISPR/Cas9 nanosystem for sono-immunometabolic cancer therapy. Nat. Commun. 13, 7903 (2022).
Shirley, J. L., de Jong, Y. P., Terhorst, C. & Herzog, R. W. Immune responses to viral gene therapy vectors. Mol. Ther. 28, 709–722 (2020).
Ramamoorth, M. & Narvekar, A. Non viral vectors in gene therapy — an overview. J. Clin. Diagn. Res. 9, GE01–GE06 (2015).
Wells, D. J. Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther. 11, 1363–1369 (2004).
Wang, M. et al. Sonoporation-induced cell membrane permeabilization and cytoskeleton disassembly at varied acoustic and microbubble-cell parameters. Sci. Rep. 8, 3885 (2018).
Landwehr, G. M. et al. Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device. Biomicrofluidics 12, 054109 (2018).
Gu, B., Posfai, E. & Rossant, J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat. Biotechnol. 36, 632–637 (2018).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Sawyer, G. J. et al. Cardiovascular function following acute volume overload for hydrodynamic gene delivery to the liver. Gene Ther. 14, 1208–1217 (2007).
Tanihara, F. et al. Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Anim. Sci. J. 90, 55–61 (2019).
Cai, J., Huang, S., Yi, Y. & Bao, S. Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells. J. Int. Med. Res. 47, 2199–2206 (2019).
Anderson, C. D. et al. Non-viral in vivo cytidine base editing in hepatocytes using focused ultrasound targeted microbubbles. Mol. Ther. Nucleic Acids 33, 733–737 (2023).
Wang, Y. et al. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Sci. Rep. 5, 8256 (2015).
Raveux, A., Vandormael-Pournin, S. & Cohen-Tannoudji, M. Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote. Sci. Rep. 7, 42661 (2017).
Hashimoto, M. & Takemoto, T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci. Rep. 5, 11315 (2015).
Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 25, 1234–1257 (2018).
Hur, J. & Chung, A. J. Microfluidic and nanofluidic intracellular delivery. Adv. Sci. 8, e2004595 (2021).
Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).
Guan, Y. et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol. Med. 8, 477–488 (2016).
Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).
Abe, T., Inoue, K. I., Furuta, Y. & Kiyonari, H. Pronuclear microinjection during S-phase increases the efficiency of CRISPR-Cas9-assisted knockin of large DNA donors in mouse zygotes. Cell Rep. 31, 107653 (2020).
Seki, A. & Rutz, S. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J. Exp. Med. 215, 985–997 (2018).
Pavlovic, K. et al. Using gene editing approaches to fine-tune the immune system. Front. Immunol. 11, 570672 (2020).
Wu, C. M. et al. Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins. J. Immunol. Methods 457, 33–40 (2018).
Moffett, H. F. et al. B cells engineered to express pathogen-specific antibodies protect against infection. Sci. Immunol. 4, eaax0644 (2019).
Huang, R. S., Lai, M. C. & Lin, S. Ex vivo expansion and CRISPR-Cas9 genome editing of primary human natural killer cells. Curr. Protoc. 1, e246 (2021).
Rautela, J., Surgenor, E. & Huntington, N. Efficient genome editing of human natural killer cells by CRISPR RNP. Preprint at bioRxiv https://doi.org/10.1101/406934 (2018).
Zhu, H. et al. Metabolic reprograming via deletion of CISH in human iPSC-derived NK cells promotes in vivo persistence and enhances anti-tumor activity. Cell Stem Cell 27, 224–237.e6 (2020).
DiTommaso, T. et al. Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo. Proc. Natl Acad. Sci. USA 115, E10907–E10914 (2018).
Murphy, K. R. et al. High-frequency irreversible electroporation brain tumor ablation: exploring the dynamics of cell death and recovery. Bioelectrochemistry 144, 108001 (2022).
Rupp, L. J. et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci. Rep. 7, 737 (2017).
Kaminski, R. et al. Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing. Sci. Rep. 6, 22555 (2016).
Kotterman, M. A., Chalberg, T. W. & Schaffer, D. V. Viral vectors for gene therapy: translational and clinical outlook. Annu. Rev. Biomed. Eng. 17, 63–89 (2015).
Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).
Naso, M. F., Tomkowicz, B., Perry, W. L. III & Strohl, W. R. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334 (2017).
Nyberg, W. A. et al. An evolved AAV variant enables efficient genetic engineering of murine T cells. Cell 186, 446–460.e19 (2023).
Li, A. et al. A self-deleting AAV-CRISPR system for in vivo genome editing. Mol. Ther. Methods Clin. Dev. 12, 111–122 (2019).
Schiwon, M. et al. One-vector system for multiplexed CRISPR/Cas9 against hepatitis B virus cccDNA utilizing high-capacity adenoviral vectors. Mol. Ther. Nucleic Acids 12, 242–253 (2018).
Wang, D. et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).
Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).
Cheong, T. C., Compagno, M. & Chiarle, R. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system. Nat. Commun. 7, 10934 (2016).
Voss, J. E. et al. Reprogramming the antigen specificity of B cells using genome-editing technologies. eLife 8, e42995 (2019).
Hung, K. L. et al. Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells. Mol. Ther. 26, 456–467 (2018).
Johnson, M. J., Laoharawee, K., Lahr, W. S., Webber, B. R. & Moriarity, B. S. Engineering of primary human B cells with CRISPR/Cas9 targeted nuclease. Sci. Rep. 9, 12144 (2018).
Li, C. et al. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9. J. Gen. Virol. 96, 2381–2393 (2015).
Sutlu, T. et al. Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy. Hum. Gene Ther. 23, 1090–1100 (2012).
Jo, D. H. et al. Simultaneous engineering of natural killer cells for CAR transgenesis and CRISPR-Cas9 knockout using retroviral particles. Mol. Ther. Methods Clin. Dev. 29, 173–184 (2023).
Pomeroy, E. J. et al. A genetically engineered primary human natural killer cell platform for cancer immunotherapy. Mol. Ther. 28, 52–63 (2020).
Jost, M. et al. CRISPR-based functional genomics in human dendritic cells. eLife 10, e65856 (2021).
Dufait, I. et al. Retroviral and lentiviral vectors for the induction of immunological tolerance. Scientifica 2012, 694137 (2012).
Aguado, B. A., Grim, J. C., Rosales, A. M., Watson-Capps, J. J. & Anseth, K. S. Engineering precision biomaterials for personalized medicine. Sci. Transl. Med. 10, eaam8645 (2018).
Behzadi, S. et al. Cellular uptake of nanoparticles: journey inside the cell. Chem. Soc. Rev. 46, 4218–4244 (2017).
Madigan, V., Zhang, F. & Dahlman, J. E. Drug delivery systems for CRISPR-based genome editors. Nat. Rev. Drug Discov. 22, 875–894 (2023).
Barua, S. & Mitragotri, S. Challenges associated with penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects. Nano Today 9, 223–243 (2014).
Luo, Y. L. et al. Macrophage-specific in vivo gene editing using cationic lipid-assisted polymeric nanoparticles. ACS Nano 12, 994–1005 (2018).
Akinc, A. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 14, 1084–1087 (2019).
Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020).
Schoenmaker, L. et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int. J. Pharm. 601, 120586 (2021).
Hassett, K. J. et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol. Ther. Nucleic Acids 15, 1–11 (2019).
Suzuki, Y. & Ishihara, H. Difference in the lipid nanoparticle technology employed in three approved siRNA (patisiran) and mRNA (COVID-19 vaccine) drugs. Drug Metab. Pharmacokinet. 41, 100424 (2021).
Tenchov, R., Bird, R., Curtze, A. E. & Zhou, Q. Lipid nanoparticles-from liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano https://doi.org/10.1021/acsnano.1c04996 (2021).
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).
Wang, M. et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl Acad. Sci. USA 113, 2868–2873 (2016).
Wei, T., Cheng, Q., Min, Y.-L., Olson, E. N. & Siegwart, D. J. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat. Commun. 11, 3232 (2020).
Miller, J. B. et al. Non‐viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co‐delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Ed. 56, 1059–1063 (2017).
Liu, J. et al. Fast and efficient CRISPR/Cas9 genome editing in vivo enabled by bioreducible lipid and messenger RNA nanoparticles. Adv. Mater. 31, 1902575 (2019).
Chen, K. et al. Engineering self-deliverable ribonucleoproteins for genome editing in the brain. Nat. Commun. 15, 1727 (2024).
Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).
Aksoy, Y. A. et al. Spatial and temporal control of CRISPR-Cas9-mediated gene editing delivered via a light-triggered liposome system. ACS Appl. Mater. Interfaces 12, 52433–52444 (2020).
Cho, E. Y. et al. Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes. J. Nanobiotechnol. 17, 19 (2019).
Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 1078–1094 (2021).
Liu, S. et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat. Mater. 20, 701–710 (2021).
Lipid nanoparticle-enabled gene editing in the lung via inhalation. Nat. Biotechnol. 41, 1394–1395 (2023).
Qiu, M. et al. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc. Natl Acad. Sci. USA 118, e2020401118 (2021).
Rosenblum, D. et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci. Adv. 6, eabc9450 (2020).
Sago, C. D. et al. High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proc. Natl Acad. Sci. USA 115, E9944–E9952 (2018).
Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018).
Gautam, M. et al. Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina. Nat. Commun. 14, 6468 (2023).
Gillmore, J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).
Longhurst, H. J. et al. CRISPR-Cas9 in vivo gene editing of KLKB1 for hereditary angioedema. N. Engl. J. Med. 390, 432–441 (2024).
Gao, Q. et al. Therapeutic potential of CRISPR/Cas9 gene editing in engineered T-cell therapy. Cancer Med. 8, 4254–4264 (2019).
Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).
Kozma, G. T., Shimizu, T., Ishida, T. & Szebeni, J. Anti-PEG antibodies: properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals. Adv. Drug Deliv. Rev. 154–155, 163–175 (2020).
Yasuda, S. et al. Comparison of the type of liposome involving cytokine production induced by non-CpG lipoplex in macrophages. Mol. Pharm. 7, 533–542 (2020).
Inglut, C. T. et al. Immunological and toxicological considerations for the design of liposomes. Nanomaterials 10, 190 (2020).
Chen, S. P. & Blakney, A. K. Immune response to the components of lipid nanoparticles for ribonucleic acid therapeutics. Curr. Opin. Biotechnol. 85, 103049 (2024).
Machtakova, M., Therien-Aubin, H. & Landfester, K. Polymer nano-systems for the encapsulation and delivery of active biomacromolecular therapeutic agents. Chem. Soc. Rev. 51, 128–152 (2022).
Emami, M. R. et al. Polyrotaxane nanocarriers can deliver CRISPR/Cas9 plasmid to dystrophic muscle cells to successfully edit the DMD gene. Adv. Ther. 2, 1900061 (2019).
Gao, X. et al. Hyperbranched poly (β-amino ester) based polyplex nanopaticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer. J. Control. Release 321, 654–668 (2020).
Li, M. et al. Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention. Nano Res. 11, 6270–6282 (2018).
Xiu, K. et al. Delivery of CRISPR/Cas9 plasmid DNA by hyperbranched polymeric nanoparticles enables efficient gene editing. Cells https://doi.org/10.3390/cells12010156 (2022).
Siemer, S. et al. Targeting cancer chemotherapy resistance by precision medicine-driven nanoparticle-formulated cisplatin. ACS Nano 15, 18541–18556 (2021).
Xie, R. et al. pH-responsive polymer nanoparticles for efficient delivery of Cas9 ribonucleoprotein with or without donor DNA. Adv. Mater. 34, e2110618 (2022).
Zhao, L. et al. HSP70-promoter-driven CRISPR/Cas9 system activated by reactive oxygen species for multifaceted anticancer immune response and potentiated immunotherapy. ACS Nano 16, 13821–13833 (2022).
Wang, R. et al. Synthesis and gene delivery of poly(amido amine)s with different branched architecture. Biomacromolecules 11, 489–495 (2010).
Kang, Y. K. et al. Nonviral genome editing based on a polymer-derivatized CRISPR nanocomplex for targeting bacterial pathogens and antibiotic resistance. Bioconjug. Chem. 28, 957–967 (2017).
Timin, A. S. et al. Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers. Nanomedicine 14, 97–108 (2018).
Sun, W. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. 54, 12029–12033 (2015).
Yue, H., Zhou, X., Cheng, M. & Xing, D. Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing. Nanoscale 10, 1063–1071 (2018).
Hryhorowicz, M. et al. Improved delivery of CRISPR/Cas9 system using magnetic nanoparticles into porcine fibroblast. Mol. Biotechnol. 61, 173–180 (2019).
Rogers, G. L. & Cannon, P. M. Genome edited B cells: a new frontier in immune cell therapies. Mol. Ther. 29, 3192–3204 (2021).
Perrin, S. & Magill, M. The inhibition of CD40/CD154 costimulatory signaling in the prevention of renal transplant rejection in nonhuman primates: a systematic review and meta analysis. Front. Immunol. 13, 861471 (2022).
Zhang, Y. et al. In situ repurposing of dendritic cells with CRISPR/Cas9-based nanomedicine to induce transplant tolerance. Biomaterials 217, 119302 (2019).
Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat. Biotechnol. 38, 44–49 (2020).
Murphy, C. J. et al. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc. Chem. Res. 41, 1721–1730 (2008).
Liu, J. et al. Dual-responsive core-shell tecto dendrimers enable efficient gene editing of cancer cells to boost immune checkpoint blockade therapy. ACS Appl. Mater. Interfaces 15, 12809–12821 (2023).
Huang, L. et al. A cancer cell membrane-derived biomimetic nanocarrier for synergistic photothermal/gene therapy by efficient delivery of CRISPR/Cas9 and gold nanorods. Adv. Healthc. Mater. 11, e2201038 (2022).
Lee, Y. W. et al. In vivo editing of macrophages through systemic delivery of CRISPR-Cas9-ribonucleoprotein-nanoparticle nanoassemblies. Adv. Ther. 2, 1900041 (2019).
Rodriguez-Izquierdo, I. et al. Gold nanoparticles crossing blood-brain barrier prevent HSV-1 infection and reduce herpes associated amyloid-βsecretion. J. Clin. Med. 9, 155 (2020).
Johnsen, K. B. et al. Modulating the antibody density changes the uptake and transport at the blood-brain barrier of both transferrin receptor-targeted gold nanoparticles and liposomal cargo. J. Control. Release 295, 237–249 (2019).
Jain, P. K., Huang, X., El-Sayed, I. H. & El-Sayed, M. A. Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine. Acc. Chem. Res. 41, 1578–1586 (2008).
Wang, P. et al. Thermo-triggered release of CRISPR-Cas9 system by lipid-encapsulated gold nanoparticles for tumor therapy. Angew. Chem. Int. Ed. 57, 1491–1496 (2018).
Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).
Mout, R. et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11, 2452–2458 (2017).
Lee, Y. W. et al. In vivo editing of macrophages through systemic delivery of CRISPR‐Cas9‐ribonucleoprotein‐nanoparticle nanoassemblies. Adv. Ther. 2, 1900041 (2019).
Willingham, S. B. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl Acad. Sci. USA 109, 6662–6667 (2012).
Alsaiari, S. K. et al. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. J. Am. Chem. Soc. 140, 143–146 (2018).
Alyami, M. Z. et al. Cell-type-specific CRISPR/Cas9 delivery by biomimetic metal organic frameworks. J. Am. Chem. Soc. 142, 1715–1720 (2020).
Pan, Y. et al. Near-infrared upconversion–activated CRISPR-Cas9 system: a remote-controlled gene editing platform. Sci. Adv. 5, eaav7199 (2019).
Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).
Rouet, R. et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. J. Am. Chem. Soc. 140, 6596–6603 (2018).
Zhang, Z. et al. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat. Biotechnol. 42, 305–315 (2023).
Foss, D. V. et al. Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nat. Biomed. Eng. 7, 647–660 (2023).
Ikwuagwu, B. & Tullman-Ercek, D. Virus-like particles for drug delivery: a review of methods and applications. Curr. Opin. Biotechnol. 78, 102785 (2022).
Hamilton, J. R. et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Rep. 35, 109207 (2021).
Kingwell, K. First CRISPR therapy seeks landmark approval. Nat. Rev. Drug Discov. 22, 339–341 (2023).
Rothgangl, T. et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotechnol. 39, 949–957 (2021).
Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).
Acknowledgements
S.K.A. thanks King Abdulaziz City of Science and Technology for Ibn Khaldun (IBK) fellowship support. M.K. thanks the Bodossaki Foundation for fellowship support. G.L. thanks the US National Science Foundation graduate research fellowship (DEG 2146752). The authors thank R. Wilson, UC Berkeley, for the feedback and comments.
Author information
Authors and Affiliations
Contributions
S.K.A., B.D. and B.E. contributed equally to the manuscript and conducted the initial literature research and, in consultation with A.J., outlined the general manuscript format. B.D., S.K.A. and B.E. wrote the manuscript draft, with contributions from M.K. and G.L. in the chemical vector section, and X.W., L.Z. and M.C. in the literature research for ex vivo delivery. X.Y. contributed to Fig. 4. B.E., S.K.A., R.L. and A.J. contributed to the manuscript revision. All authors reviewed and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
A.J. receives licensing fees from patents in which she was an inventor, and she invested in, consults for (or was on Scientific Advisory Boards or Boards of Directors), lectured at (and received a fee), or conducts sponsored research at MIT, for which she was not paid, for the following entities: The Estée Lauder Companies, Moderna Therapeutics, OmniPulse Biosciences, Particles for Humanity, SiO2 Materials Science, and VitaKey. For a list of entities with which R.L. is or has been recently involved with or had been compensated or uncompensated by, see Supplementary information. All other authors declare no competing interests.
Peer review
Peer review information
Nature Reviews Materials thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Alsaiari, S.K., Eshaghi, B., Du, B. et al. CRISPR–Cas9 delivery strategies for the modulation of immune and non-immune cells. Nat Rev Mater 10, 44–61 (2025). https://doi.org/10.1038/s41578-024-00725-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41578-024-00725-7
This article is cited by
-
Advancing cancer gene therapy: the emerging role of nanoparticle delivery systems
Journal of Nanobiotechnology (2025)
