Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cox DBT, Platt RJ, Zhang F . Therapeutic genome editing: prospects and challenges. Nat Med 2015; 21: 121–131.
Carroll D . Genome engineering with targetable nucleases. Annu Rev Biochem 2014; 83: 409–439.
Gersbach CA . Genome engineering: the next genomic revolution. Nat Meth 2014; 11: 1009–1011.
Maggio I, Gonçalves MAFV . Genome editing at the crossroads of delivery, specificity, and fidelity. Trends Biotech 2015; 33: 280–291.
Gaj T, Gersbach CA, Barbas CF 3rd . ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotech 2013; 31: 397–405.
Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014; 370: 901–910.
Poirot L, Philip B, Schiffer-Mannioui C, Le Clerre D, Chion-Sotinel I, Derniame S et al. Multiplex genome-edited T-cell manufacturing platform for ‘Off-the-Shelf’ adoptive T-cell immunotherapies. Cancer Res 2015; 75: 3853–3864.
Kirchner M, Schneider S . CRISPR-Cas: from the bacterial adaptive immune system to a versatile tool for genome engineering. Angew Chem Int Ed 2015; 54: 13508–13514.
Doudna JA, Charpentier E . The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346: 6213.
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014; 343: 84–87.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819–823.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337: 816–821.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al. RNA-guided human genome engineering via Cas9. Science 2013; 339: 823–826.
Hsu PD, Lander ES, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 15: 1262–1278.
Mali P, Esvelt KM, Church GM . Cas9 as a versatile tool for engineering biology. Nat Meth 2013; 10: 957–963.
Sander JD, Joung JK . CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotech 2014; 32: 347–355.
Jasin M, Haber JE . The democratization of gene editing: insights from site-specific cleavage and double-strand break repair. DNA Repair 2016; 44: 6–16.
Keener AB . Delivering the goods: scientists seek a way to make CRISPR-Cas gene editing more targeted. Nat Med 2015; 21: 1239–1241.
Nelson CE, Gersbach CA . Engineering delivery vehicles for genome editing. Annu Rev Chem Biomol Eng 2016; 7: 637–662.
Gori JL, Hsu PD, Maeder ML, Shen S, Welstead GG, Bumcrot D . Delivery and specificity of CRISPR/Cas9 genome editing technologies for human gene therapy. Hum Gene Ther 2015; 26: 443–451.
Li L, He Z-Y, Wei X-W, Gao G-P, Wei Y-Q . Challenges in CRISPR/Cas9 delivery: potential roles of nonviral vectors. Hum Gene Ther 2015; 26: 452–462.
LaFountaine JS, Fathe K, Smyth HDC . Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm 2015; 494 (1): 180–194.
Ain QU, Chung JY, Kim Y-H . Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release 2015; 205: 120–127.
Holkers M, Cathomen T, Gonçalves MAFV . Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells. Methods 2014; 69: 179–187.
Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 2011; 475: 217–221.
Choi JG, Dang Y, Abraham S, Ma H, Zhang J, Guo H et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther 2016; 23: 627–633.
Cai Y, Bak RO, Mikkelsen JG . Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. eLife 2014; 3: e01911.
Chen X, Goncalves MAFV . Engineered viruses as genome editing devices. Mol Ther 2016; 44: 447–457.
Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015; 520: 186–191.
Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotech 2015; 208: 44–53.
Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotech 2015; 33: 73–80.
Kim S, Kim D, Cho SW, Kim J, Kim J-S . Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 2014; 24: 1012–1019.
Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci USA 2015; 112: 10437–10442.
Suda T, Liu D . Hydrodynamic gene delivery: its principles and applications. Mol Ther 2007; 15: 2063–2069.
Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotech 2014; 32 (6): 551–553.
Bijarnia S, Puri RD, Ruel J, Gray GF, Jenkinson L, Verma IC . Tyrosinemia type I–diagnostic issues and prenatal diagnosis. Indian J Pediatr 2006; 73 (2): 163–165.
Nakase I, Akita H, Kogure K, Gräslund A, Langel Ü, Harashima H et al. Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides. Acc Chem Res 2012; 45: 1132–1139.
Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S et al. Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs. Cell Regen 2013; 2: 5.
Liu J, Gaj T, Patterson JT, Sirk SJ, Barbas Iii CF . Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS One 2014; 9: e85755.
Ramakrishna S, Kwaku Dad A-B, Beloor J, Gopalappa R, Lee S-K, Kim H . Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 2014; 24: 1020–1027.
Rizzuti M, Nizzardo M, Zanetta C, Ramirez A, Corti S . Therapeutic applications of the cell-penetrating HIV-1 Tat peptide. Drug Discov Today 2015; 20: 76–85.
Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG . Non-viral vectors for gene-based therapy. Nat Rev Genet 2014; 15: 541–555.
Gu Z, Biswas A, Zhao M, Tang Y . Tailoring nanocarriers for intracellular protein delivery. Chem Soc Rev 2011; 40: 3638–3655.
Peer D, Karp JM, Hong S, FaroKHzad OC, Margalit R, Langer R . Nanocarriers as an emerging platform for cancer therapy. Nat Nanotech 2007; 2: 751–760.
Matsumura Y, Maeda H . A new concept for macromolecular therapeutics in cancer-chemotherapy - mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986; 46: 6387–6392.
Frokjaer S, Otzen DE . Protein drug stability: a formulation challenge. Nat Rev Drug Discov 2005; 4: 298–306.
Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, Augustus S et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435: 646–651.
Cradick TJ, Keck K, Bradshaw S, Jamieson AC, McCaffrey AP . Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol Ther 2010; 18: 947–954.
Hu Z, Ding W, Zhu D, Yu L, Jiang X, Wang X et al. TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy. J Clin Invest 2015; 125: 425–436.
Mahiny AJ, Dewerth A, Mays LE, Alkhaled M, Mothes B, Malaeksefat E et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat Biotech 2015; 33: 584–586.
Wang M, Zuris JA, Meng F, Rees H, Sun S, Deng P et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci USA 2016; 113: 2868–2873.
Wang M, Alberti K, Varone A, Pouli D, Georgakoudi I, Xu Q . Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles. Adv Health Mater 2014; 3: 1398–1403.
Altinoglu S, Wang M, Xu Q . Combinatorial library strategies for synthesis of cationic lipid-like nanoparticles and their potential medical applications. Nanomedicine 2015; 10: 643–657.
Wang M, Sun S, Neufeld CI, Perez-Ramirez B, Xu Q . Reactive oxygen species-responsive protein modification and its intracellular delivery for targeted cancer therapy. Angew Chem Int Ed 2014; 53: 13444–13448.
Wang M, Alberti K, Sun S, Arellano C, Xu Q . Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy. Angew Chem Int Ed 2014; 53: 2893–2898.
Wang M, Sun S, Alberti K, Xu Q . A combinatorial library of unsaturated lipidoids for efficient intracellular gene delivery. ACS Synth Biol 2012; 1: 403–407.
Sun S, Wang M, Knupp S, Soto-Feliciano Y, Hu X, Kaplan D et al. Combinatorial library of lipidoids for in vitro DNA delivery. Bioconjugate Chem 2012; 23: 135–140.
Sun S, Wang M, Alberti K, Choy A, Xu Q . DOPE facilitates quaternized lipidoids (QLDs) for in vitro DNA delivery. Nanomedicine 2013; 9: 849–854.
Sun W, Ji W, Hall JM, Hu Q, Wang C, Beisel CL et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing. Angew Chem Int Ed 2015; 54: 12029–12033.
D’Astolfo Diego S, Pagliero Romina J, Pras A, Karthaus Wouter R, Clevers H, Prasad V et al. Efficient intracellular delivery of native proteins. Cell 2015; 161: 674–690.
Yin H, Song C-Q, Dorkin JR, Zhu LJ, Li Y, Wu Q et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotech 2016; 34: 328–333.
The authors declare no conflict of interest.
This research is supported by the National Science Foundation grant (DMR 1452122) awarded to Dr Qiaobing Xu
About this article
Cite this article
Wang, M., Glass, Z. & Xu, Q. Non-viral delivery of genome-editing nucleases for gene therapy. Gene Ther 24, 144–150 (2017). https://doi.org/10.1038/gt.2016.72
Efficient construction of a stable linear gene based on a TNA loop modified primer pair for gene delivery
Chemical Communications (2020)
Hydrophobically modified carbon dots as a multifunctional platform for serum-resistant gene delivery and cell imaging
Biomaterials Science (2020)
Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications
Advanced Materials (2020)
A Lactose‐Derived CRISPR/Cas9 Delivery System for Efficient Genome Editing In Vivo to Treat Orthotopic Hepatocellular Carcinoma
Advanced Science (2020)
Journal of Materials Chemistry B (2020)