Delivering CRISPR: a review of the challenges and approaches

Figures & data

Figure 1. Following formation of a double stranded break (DSB), endogenous DNA repair can occur by (A) non-homologous end joining (NHEJ) resulting in random indels, or by (B) homology-directed repair (HDR) which uses a template DNA strand for precise repair.

Figure 2. Products of site-specific nuclease-based gene editing: (A) gene knockout, (B) gene deletion, (C) gene correction, and (D) gene addition.

Figure 3. Site-specific endonucleases with programable DNA-binding protein domains: (A) zinc finger nucleases (ZFNs) and (B) transcription activator-like effector nucleases (TALENs).

Figure 4. Biology of the type II CRISPR/Cas system. (A) Genomic representation of CRISPR/Cas9 along with relevant transcription/translation products. (B) Engineered CRISPR/Cas9 for site-specific gene editing (sgRNA:Cas9). Grey arrows indicate sites of single-stranded nucleotide breaks.

Table 1. CRISPR delivery vehicles and their common features. Relatively difficulty is a subjective measure of how difficult the delivery vehicle is to utilize overall on a four-point scale, where one point is ‘few reagents, facile kit provided’ and four points is ‘requires expert in field with significant experimental experience’.

Delivery vehicle	Composition	Most common cargo	Capacity	Advantages	Limitations	Ease of use	Text refs
Microinjection	Needle	DNA plasmid; mRNA (Cas9 + sgRNA); Protein (RNP)	nM levels of Cas9 and sgRNA	Guaranteed delivery into cell of interest	Time-consuming; difficult; generally in vitro only	****	Yang et al. (2013), Horii et al. (2014), Chuang et al. (2017), Nakagawa et al. (2015), Crispo et al. (2015), Raveux et al. (2017), Sato et al. (2015), Ma et al. (2014), Niu et al. (2014), Wu et al. (2013), Long et al. (2014), Ross (1995)
Electroporation; nucleofection	Electric current	DNA plasmid; mRNA (Cas9 + sgRNA)	nM levels of Cas9 and sgRNA	Delivery to cell population; well-known technique	Generally in vitro only; some cells not amenable	*	Hashimoto & Takemoto (2015), Chen et al. (2016), Qin et al. (2015), Matano et al. (2015), Paquet et al. (2016), Ousterout et al. (2015), Schumann et al. (2015), Wu et al. (2015), Ye et al. (2014), Choi et al. (2014), Wang et al. (2014), Zuckermann et al. (2015), Kim et al. (2014)
Hydrodynamic delivery	High-pressure injection	DNA plasmid; Protein (RNP)	nM levels of Cas9 and sgRNA	Virus-free; low cost; ease	Non-specific; traumatic to tissues	**	Yin et al. (2014), Guan et al. (2016), Xue et al. (2014), Lin et al. (2014), Zhen et al. (2015), Dong et al. (2015)
Adeno-associated virus (AAV)	Non-enveloped, ssDNA	DNA plasmid	<5kb nucleic acid	Minimal immunogenicity	Low capacity	***	Yang et al. (2013), Long et al. (2016), Carroll et al. (2016), Platt et al. (2014), Hung et al. (2016), Swiech et al. (2015), Chew et al. (2016), Truong et al. (2015), Ran et al. (2015), Nelson et al. (2016), Tabebordbar et al. (2016), Esvelt et al. (2013)
Adenovirus	Non-enveloped, dsDNA	DNA plasmid	8kb nucleic acid	High efficiency delivery	Inflammatory response; difficult scaled production	***	Voets et al. (2017), Maddalo et al. (2014), Wang et al. (2015), Ding et al. (2014), Maggio et al. (2016), Li et al. (2015), Cheng et al. (2014)
Lentivirus	Enveloped, RNA	DNA plasmid	∼10kb, up to 18 kb nucleic acid	Persistent gene transfer	Prone to gene rearrangement; transgene silencing	***	Shalem et al. (2014), Wang et al. (2014), Naldini et al. (1996), Kabadi et al. (2014), Heckl et al. (2014); Roehm et al. (2016), Koike-Yusa et al. (2014), Ma et al. (2015), Zhang et al. (2016), Platt et al. (2014)
Lipid nanoparticles/ liposomes/lipoplexes	Natural or synthetic lipids or polymers	mRNA (Cas9 + sgRNA); Protein (RNP)	nM levels of Cas9 and sgRNA	Virus-free; simple manipulation; low cost	Endosomal degradation of cargo; specific cell tropism	**	Yin et al. (2016), Wang et al. (2016), Zuris et al. (2015), Horii et al. (2013), Sakuma et al. (2014), Schwank et al. (2013), Liu et al. (2014), Liang et al. (2015), Kennedy et al. (2014), Miller et al. (2017), Ebina et al. (2013)
Cell-penetrating peptides (CPPs)	Short amino acid sequences	Protein (RNP)	nM levels of Cas9 and sgRNA	Virus-free; can deliver intact RNP	Variable penetrating efficiency	**	Ramakrishna et al. (2014), Axford et al. (2017)
DNA nanoclew	DNA spheroid	Protein (RNP)	nM levels of Cas9 and sgRNA	Virus-free	Modifications for template DNA needed	****	Sun et al. (2015), Sun et al. (2014)
Gold nanoparticles (AuNPs)	Cationic arginine-coated AuNP	Protein (RNP)	nM levels of Cas9 and sgRNA	Inert; membrane-fusion-like delivery	Nonspecific inflammatory response	**	Mout et al. (2017), Lee et al. (2017)
iTOP	Hyperosmlality + transduction compound	Protein (RNP)	nM levels of Cas9 and sgRNA	Virus-free; high-efficiency	Non-specific; no in vivo use yet reported	***	D'Astolfo et al. (2015)
SLO	Bacterial pore-forming toxin	∼100kDa proteins and complexes	Unknown for CRISPR	Reversible pore formation; no impact on cell viability	Not yet proven with CRISPR	***	Sierig et al. (2003), Walev et al. (2001), Brito et al. (2008), Teng et al. (2017)
MENDs	Poly-lysine core, lipid coating, CPP decoration	Nucleic acids	Unknown for CRISPR	Customizable; readily modified for precise delivery	Not yet proven with CRISPR	****	Kogure et al. (2004), Nakamura et al. (2012)
Lipid-coated mesoporous silica NPs	Mesoporous Si coated with lipid	Small molecules and short RNA sequences	Unknown for CRISPR	Inert; easy modification with targeting moieties	Not yet proven with CRISPR	***	Liu et al. (2009), Du et al. (2014), Durfee et al. (2016), Gonzalez Porras et al. (2016), Mackowiak et al. (2013), Su et al. (2017), Wang et al. (2013)
Inorganic NPs	NPs of various compositions (carbon, silica)	Large proteins, nucleic acids	Unknown for CRISPR	Inert; used for similar applications	Not yet proven with CRISPR	**	Bates & Kostarelos (2013), Luo et al. (2014), Luo & Saltzman (2000)

Figure 5. Physical methods for delivery of CRISPR. (A) Microinjection disrupting two genes (Ppar-γ and Rag1) in Cynomolgus monkeys from a single injection into one-cell-stage embryos. Photographs of Founder Monkeys A and B, PCR products of the targeted loci from genomic DNA of A and B, and a control wild-type Cynomolgus monkey (Con). Adapted with permission from Nui et al. (2014). Copyright 2014 Elsevier Inc. (B) Electroporation delivery of CRISPR RNP targeting genes impacting mice coat color (Tyr) followed by transfer to pseudopregnant mothers. Bar plot quantifies coat color phenotypes generated from microinjection and electroporation at 1 ms pulse length and 3 ms pulse length. Adapted with permission from Chen et al. (2016). Copyright 2016 American Society for Biochemistry and Molecular Biology. (C) Hydrodynamic injection of CRISPR into mice results in liver-specific targeting (see bioluminescence image of hydrodynamically injected luciferase plasmid), generating indel mutation of two tumor suppressor genes and oncogenes. The development of liver tumors can be seen in the hematoxylin and eosin (H&E) and cytokeratin 19 (Ck19)-stained micrographs. Adapted with permission from Xue et al. (2014). Copyright 2014 Macmillan Publishers Ltd: Nature.

Figure 6. Viral vector methods for delivery of CRISPR. (A) AAV delivery of Cas9 and sgRNAs disrupting mutations in the Dmd gene in adult mdx mice, resulting in improvement of muscle biochemistry and function. Adapted with permission from Long et al. (2016). Copyright 2016 American Association for the Advancement of Science. (B) AAV intratracheal instillation delivery of sgRNAs in Cre-dependent Cas9 knock-in mice, resulting in lung adenocarcinoma (EGFP-positive tumors). Adapted with permission from Platt et al. (2016). Copyright 2014 Elsevier Inc. (C) A split Cas9 system in which the Cas9 C-terminal is packaged into one AAV vector and the Cas9 N-terminal is packaged into a second AAV vector. Reconstitution results in a fully functioning Cas9. Reprinted from Truong et al. (2015). Copyright 2014 The Authors (CC BY license). (D) AdV delivery of Cas9 and sgRNA targeting the Pten gene in mouse liver resulting in Pten mutation (see arrows by gel), and massive hepatomegaly and features of NASH in infected livers. Immunohistochemistry shows loss of Pten staining (arrows) one month after AdV infection; H&E stained micrographs show sections of steatosis (lipid accumulation, arrows) four months post infection. Adapted with permission from Wang et al., 2015. Copyright 2015 Mary Ann Liebert, Inc. Publishers. (E) Schematic of a lentivirus and CRISPR-based gene library functional screen used to identify the genes essential for West-Nile-virus-induced cell death. Reprinted from Ma et al. (2015). Copyright 2015 The Authors (CC BY license).

Figure 7. Lipid nanoparticle and polyplex delivery of CRISPR. (A) Combining bioreducible lipid nanoparticles and anionic Cas9:sgRNA complexes drives the electrostatic self-assembly of nanoparticles (see TEM micrograph of 3-O14B/Cas9:sgRNA nanoparticles) for potent protein delivery and genome editing. Adapted with permission from Wang et al. (2016). Copyright 2017 National Academy of Sciences. (B) Microinjection of PEI with Cas9- and sgRNA-encoding plasmid DNA into mouse brain directed against the Ptch1 locus to generate a malignant brain tumor model. Compare the wild type and Trp53± H&E stained micrographs (arrows indicate small lesions encompassing only one cerebellar folium) with the tumor from the Trp53^−/− condition (MB = medulloblastoma). Adapted from Zuckermann et al. (2015). Copyright 2015 The Authors (CC BY license).

Figure 8. Nanomaterial delivery vehicles for CRISPR delivery. (A) DNA ‘nanoclew’ loaded with Cas9:sgRNA RNP via Watson-Crick base pairing, followed by coating with PEI to improve endosomal escape. Reprinted with permission from Sun et al. (2015). Copyright 2015 John Wiley & Sons. (B) Arginine-modified gold nanoparticles (ArgNPs, positively charged) interact with multiple Cas9:sgRNA RNPs engineered with an E-tag to form a local negatively charged region, forming a nanoassembly that delivers Cas9 via membrane fusion. Reprinted with permission from Mout et al. (2017). Copyright 2017 American Chemical Society. (C) Synthesis of AuNPs engineered to complex with multiple Cas9:sgRNA RNPs, followed by coating in silica and the endosomal disruptive polymer PASp(DET). Adapted with permission from Lee et al. (2017). Copyright 2017 Macmillan Publishers Ltd: Nature Biomedical Engineering.

Yang H, Wang H, Shivalila CS, et al. (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–9.

 PubMed Web of Science ®Google Scholar

Horii T, Arai Y, Yamazaki M, et al. (2014). Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep 4:4513.

 PubMed Web of Science ®Google Scholar

Chuang CK, Chen CH, Huang CL, et al. (2017). Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors. Anim Biotechnol 28:174–81.

 PubMed Web of Science ®Google Scholar

Nakagawa Y, Sakuma T, Sakamoto T, et al. (2015). Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol 15:33.

 PubMed Web of Science ®Google Scholar

Crispo M, Mulet AP, Tesson L, et al. (2015). Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PLoS One 10:e0136690.

 PubMed Web of Science ®Google Scholar

Raveux A, Vandormael-Pournin S, Cohen-Tannoudji M. (2017). Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote. Sci Rep 7:42661.

 PubMed Web of Science ®Google Scholar

Sato M, Koriyama M, Watanabe S, et al. (2015). Direct injection of CRISPR/Cas9-related mRNA into cytoplasm of parthenogenetically activated porcine oocytes causes frequent mosaicism for indel mutations. Ijms 16:17838–56.

 Web of Science ®Google Scholar

Ma Y, Shen B, Zhang X, et al. (2014). Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One 9:e89413.

 PubMed Web of Science ®Google Scholar

Niu Y, Shen B, Cui Y, et al. (2014). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–43.

 PubMed Web of Science ®Google Scholar

Wu Y, Liang D, Wang Y, et al. (2013). Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–62.

 PubMed Web of Science ®Google Scholar

Long C, McAnally JR, Shelton JM, et al. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184–8.

 PubMed Web of Science ®Google Scholar

Ross J. (1995). mRNA stability in mammalian cells. Microbiol Rev 59:423–50.

 PubMedGoogle Scholar

Hashimoto M, Takemoto T. (2015). Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5:11315.

 PubMed Web of Science ®Google Scholar

Chen S, Lee B, Lee AY, et al. (2016). Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291:14457–67.

 PubMed Web of Science ®Google Scholar

Qin W, Dion SL, Kutny PM, et al. (2015). Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200:423–30.

 PubMed Web of Science ®Google Scholar

Matano M, Date S, Shimokawa M, et al. (2015). Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 21:256–62.

 PubMed Web of Science ®Google Scholar

Paquet D, Kwart D, Chen A, et al. (2016). Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–9.

 PubMed Web of Science ®Google Scholar

Ousterout DG, Kabadi AM, Thakore PI, et al. (2015). Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun 6:6244.

 PubMed Web of Science ®Google Scholar

Schumann K, Lin S, Boyer E, et al. (2015). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci USA 112:10437–42.

 PubMed Web of Science ®Google Scholar

Wu Y, Zhou H, Fan X, et al. (2015). Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 25:67–79.

 PubMed Web of Science ®Google Scholar

Ye L, Wang J, Beyer AI, et al. (2014). Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci USA 111:9591–6.

 PubMed Web of Science ®Google Scholar

Wang T, Wei JJ, Sabatini DM, Lander ES. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–4.

 PubMed Web of Science ®Google Scholar

Zuckermann M, Hovestadt V, Knobbe-Thomsen CB, et al. (2015). Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat Commun 6:7391.

 PubMed Web of Science ®Google Scholar

Kim S, Kim D, Cho SW, et al. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–9.

 PubMed Web of Science ®Google Scholar

Yin H, Xue W, Chen S, et al. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32:551–3.

 PubMed Web of Science ®Google Scholar

Guan Y, Ma Y, Li Q, et al. (2016). CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 8:477–88.

 PubMed Web of Science ®Google Scholar

Xue W, Chen S, Yin H, et al. (2014). CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514:380–4.

 PubMed Web of Science ®Google Scholar

Lin S, Staahl BT, Alla RK, Doudna JA. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766.

 PubMed Web of Science ®Google Scholar

Zhen S, Hua L, Liu YH, et al. (2015). Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther 22:404–12.

 PubMed Web of Science ®Google Scholar

Dong C, Qu L, Wang H, et al. (2015). Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res 118:110–7.

 PubMed Web of Science ®Google Scholar

Long C, Amoasii L, Mireault AA, et al. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351:400–3.

 PubMed Web of Science ®Google Scholar

Carroll KJ, Makarewich CA, McAnally J, et al. (2016). A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9. Proc Natl Acad Sci USA 113:338–43.

 PubMed Web of Science ®Google Scholar

Platt RJ, Chen S, Zhou Y, et al. (2014). CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–55.

 PubMed Web of Science ®Google Scholar

Hung SS, Chrysostomou V, Li F, et al. (2016). AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo. Invest Ophthalmol Vis Sci 57:3470–6.

 PubMed Web of Science ®Google Scholar

Swiech L, Heidenreich M, Banerjee A, et al. (2015). In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33:102–6.

 PubMed Web of Science ®Google Scholar

Chew WL, Tabebordbar M, Cheng JK, et al. (2016). A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 13:868–74.

 PubMed Web of Science ®Google Scholar

Truong DJ, Kuhner K, Kuhn R, et al. (2015). Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res 43:6450–8.

 PubMed Web of Science ®Google Scholar

Ran FA, Cong L, Yan WX, et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–91.

 PubMed Web of Science ®Google Scholar

Nelson CE, Hakim CH, Ousterout DG, et al. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403–7.

 PubMed Web of Science ®Google Scholar

Tabebordbar M, Zhu K, Cheng JKW, et al. (2016). In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:407–11.

 PubMed Web of Science ®Google Scholar

Esvelt KM, Mali P, Braff JL, et al. (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10:1116–21.

 PubMed Web of Science ®Google Scholar

Voets O, Tielen F, Elstak E, et al. (2017). Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells. PLoS One 12:e0182974.

 PubMed Web of Science ®Google Scholar

Maddalo D, Manchado E, Concepcion CP, et al. (2014). In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516:423–7.

 PubMed Web of Science ®Google Scholar

Wang D, Mou H, Li S, et al. (2015). Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum Gene Ther 26:432–42.

 PubMed Web of Science ®Google Scholar

Ding Q, Strong A, Patel KM, et al. (2014). Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 115:488–92.

 PubMed Web of Science ®Google Scholar

Maggio I, Stefanucci L, Janssen JM, et al. (2016). Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations. Nucleic Acids Res 44:1449–70.

 PubMed Web of Science ®Google Scholar

Li C, Guan X, Du T, et al. (2015). Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9. J Gen Virol 96:2381–93.

 PubMed Web of Science ®Google Scholar

Cheng R, Peng J, Yan Y, et al. (2014). Efficient gene editing in adult mouse livers via adenoviral delivery of CRISPR/Cas9. FEBS Lett 588:3954–8.

 PubMed Web of Science ®Google Scholar

Shalem O, Sanjana NE, Hartenian E, et al. (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–7.

 PubMed Web of Science ®Google Scholar

Naldini L, Blomer U, Gallay P, et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–7.

 PubMed Web of Science ®Google Scholar

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. (2014). Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res 42:e147.

 PubMed Web of Science ®Google Scholar

Heckl D, Kowalczyk MS, Yudovich D, et al. (2014). Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol 32:941–6.

 PubMed Web of Science ®Google Scholar

Roehm PC, Shekarabi M, Wollebo HS, et al. (2016). Inhibition of HSV-1 replication by gene editing strategy. Sci Rep 6:23146.

 PubMed Web of Science ®Google Scholar

Koike-Yusa H, Li Y, Tan EP, et al. (2014). Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–73.

 PubMed Web of Science ®Google Scholar

Ma H, Dang Y, Wu Y, et al. (2015). A CRISPR-based screen identifies genes essential for West-Nile-Virus-induced cell death. Cell Rep 12:673–83.

 PubMed Web of Science ®Google Scholar

Zhang D, Li Z, Li JF. (2016). Targeted gene manipulation in plants using the CRISPR/Cas technology. J Genet Genomics = Yi Chuan Xue Bao 43:251–62.

 PubMed Web of Science ®Google Scholar

Yin H, Song CQ, Dorkin JR, et al. (2016). Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34:328–33.

 PubMed Web of Science ®Google Scholar

Wang M, Zuris JA, Meng F, et al. (2016). Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci USA 113:2868–73.

 PubMed Web of Science ®Google Scholar

Zuris JA, Thompson DB, Shu Y, et al. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80.

 PubMed Web of Science ®Google Scholar

Horii T, Tamura D, Morita S, et al. (2013). Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system. IJMS 14:19774–81.

 Web of Science ®Google Scholar

Sakuma T, Nishikawa A, Kume S, et al. (2014). Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep 4:5400.

 PubMed Web of Science ®Google Scholar

Schwank G, Koo BK, Sasselli V, et al. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13:653–8.

 PubMed Web of Science ®Google Scholar

Liu Y, Zeng Y, Liu L, et al. (2014). Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells. Nat Commun 5:5393.

 PubMed Web of Science ®Google Scholar

Liang X, Potter J, Kumar S, et al. (2015). Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53.

 PubMed Web of Science ®Google Scholar

Kennedy EM, Kornepati AV, Goldstein M, et al. (2014). Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. J Virol 88:11965–72.

 PubMed Web of Science ®Google Scholar

Miller JB, Zhang S, Kos P, et al. (2017). 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 Engl 56:1059–63.

 PubMed Web of Science ®Google Scholar

Ebina H, Misawa N, Kanemura Y, Koyanagi Y. (2013). Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3:2510.

 PubMed Web of Science ®Google Scholar

Ramakrishna S, Kwaku Dad AB, Beloor J, et al. (2014). Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–7.

 PubMed Web of Science ®Google Scholar

Axford DS, Morris DP, McMurry JL. (2017). Cell penetrating peptide-mediated nuclear delivery of Cas9 to enhance the utility of CRISPR/Cas genome editing. FASEB J 31:909.4.

 Web of Science ®Google Scholar

Sun W, Ji W, Hall JM, et al. (2015). Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew Chem Int Ed Engl 54:12029–33.

 PubMed Web of Science ®Google Scholar

Sun W, Jiang T, Lu Y, et al. (2014). Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery. J Am Chem Soc 136:14722–5.

 PubMed Web of Science ®Google Scholar

Mout R, Ray M, Yesilbag Tonga G, et al. (2017). Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11:2452–8.

 PubMed Web of Science ®Google Scholar

Lee K, Conboy M, Park HM, et al. (2017). Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 1:889–901.

 PubMed Web of Science ®Google Scholar

D'Astolfo DS, Pagliero RJ, Pras A, et al. (2015). Efficient intracellular delivery of native proteins. Cell 161:674–690.

 PubMed Web of Science ®Google Scholar

Sierig G, Cywes C, Wessels MR, Ashbaugh CD. (2003). Cytotoxic effects of streptolysin o and streptolysin s enhance the virulence of poorly encapsulated group a streptococci. Infect Immun 71:446–55.

 PubMed Web of Science ®Google Scholar

Walev I, Bhakdi SC, Hofmann F, et al. (2001). Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc Natl Acad Sci U S A 98:3185–90.

 PubMed Web of Science ®Google Scholar

Brito JL, Davies FE, Gonzalez D, Morgan GJ. (2008). Streptolysin-O reversible permeabilisation is an effective method to transfect siRNAs into myeloma cells. J Immunol Methods 333:147–55.

 PubMed Web of Science ®Google Scholar

Teng KW, Ishitsuka Y, Ren P, et al. (2017). Labeling proteins inside living cells using external fluorophores for fluorescence microscopy. Elife 6:e25460.

 PubMedGoogle Scholar

Kogure K, Moriguchi R, Sasaki K, et al. (2004). Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method. J Control Release 98:317–23.

 PubMed Web of Science ®Google Scholar

Nakamura T, Akita H, Yamada Y, et al. (2012). A multifunctional envelope-type nanodevice for use in nanomedicine: concept and applications. Acc Chem Res 45:1113–21.

 PubMed Web of Science ®Google Scholar

Liu J, Stace-Naughton A, Jiang X, Brinker CJ. (2009). Porous nanoparticle supported lipid bilayers (protocells) as delivery vehicles. J Am Chem Soc 131:1354–5.

 PubMed Web of Science ®Google Scholar

Du X, Shi B, Tang Y, et al. (2014). Label-free dendrimer-like silica nanohybrids for traceable and controlled gene delivery. Biomaterials 35:5580–90.

 PubMed Web of Science ®Google Scholar

Durfee PN, Lin YS, Dunphy DR, et al. (2016). Mesoporous silica nanoparticle-supported lipid bilayers (protocells) for active targeting and delivery to individual leukemia cells. ACS Nano 10:8325–45.

 PubMed Web of Science ®Google Scholar

Gonzalez Porras MA, Durfee PN, Gregory AM, et al. (2016). A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells. J Neurosci Methods 273:160–74.

 PubMed Web of Science ®Google Scholar

Mackowiak SA, Schmidt A, Weiss V, et al. (2013). Targeted drug delivery in cancer cells with red-light photoactivated mesoporous silica nanoparticles. Nano Lett 13:2576–83.

 PubMed Web of Science ®Google Scholar

Su J, Sun H, Meng Q, et al. (2017). Enhanced blood suspensibility and laser-activated tumor-specific drug release of theranostic mesoporous silica nanoparticles by functionalizing with erythrocyte membranes. Theranostics 7:523–37.

 PubMed Web of Science ®Google Scholar

Wang D, Huang J, Wang X, et al. (2013). The eradication of breast cancer cells and stem cells by 8-hydroxyquinoline-loaded hyaluronan modified mesoporous silica nanoparticle-supported lipid bilayers containing docetaxel. Biomaterials 34:7662–73.

 PubMed Web of Science ®Google Scholar

Bates K, Kostarelos K. (2013). Carbon nanotubes as vectors for gene therapy: past achievements, present challenges and future goals. Adv Drug Deliv Rev 65:2023–33.

 PubMed Web of Science ®Google Scholar

Luo GF, Chen WH, Liu Y, et al. (2014). Multifunctional enveloped mesoporous silica nanoparticles for subcellular co-delivery of drug and therapeutic peptide. Sci Rep 4:6064.

 PubMed Web of Science ®Google Scholar

Luo D, Saltzman WM. (2000). Enhancement of transfection by physical concentration of DNA at the cell surface. Nat Biotechnol 18:893–5.

 PubMed Web of Science ®Google Scholar