Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

Rouet, Romain; Thuma, Benjamin A.; Roy, Marc D.; Lintner, Nathanael G.; Rubitski, David M.; Finley, James E.; Wisniewska, Hanna M.; Mendonsa, Rima; Hirsh, Ariana; Oñate, Lorena de; Compte, Joan; McLellan, Thomas J.; Bellenger, Justin; Feng, Xidong; Varghese, Alison H.; Chrunyk, Boris A.; Borzilleri, Kris A.; Hesp, Kevin D.; Zhou, Kaihong; Ma, Nannan; Tu, Meihua; Dullea, Robert; McClure, Kim F.; Wilson, Ross C.; Liras, Spiros; Mascitti, Vincent; Doudna, Jennifer A.

doi:10.1021/jacs.8b01551

Cited by 136 publications

(122 citation statements)

References 48 publications

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“…27 This was then complexed with Cas9 ribonucleoprotein and cationic endosomal disruptive polymers. 29 For example, Cas9 proteins harbouring asialoglycoprotein receptor ligands increased the accumulation in HepG2 cells, which were highly expressing the asialoglycoprotein receptor, compared with control SKHEP cells, which express low levels of this receptor, 29 possibly due to asialoglycoprotein receptor-mediated endocytosis. 27 Local injection of this CRISPR-Gold was shown to efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice.…”

Section: The Crispr/cas9 System and Its Deliverymentioning

confidence: 99%

“…28 On the other hand, cell-type-selective delivery method has been reported, which was mediated by interactions between receptor and its specific ligand. 29 For example, Cas9 proteins harbouring asialoglycoprotein receptor ligands increased the accumulation in HepG2 cells, which were highly expressing the asialoglycoprotein receptor, compared with control SKHEP cells, which express low levels of this receptor, 29 possibly due to asialoglycoprotein receptor-mediated endocytosis. Although the in vivo efficiency remains unclear, it represents an ideal strategy for precise delivery of the Cas9 materials worthy of further investigations.…”

Section: The Crispr/cas9 System and Its Deliverymentioning

confidence: 99%

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Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9

Afolabi

Adeshakin

Sani³

et al. 2019

Immunology

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Summary Natural killer cells are potent cytotoxic lymphocytes specialized in recognizing and eliminating transformed cells, and in orchestrating adaptive anti‐tumour immunity. However, NK cells are usually functionally exhausted in the tumour microenvironment. Strategies such as checkpoint blockades are under investigation to overcome NK cell exhaustion in order to boost anti‐tumour immunity. The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene‐editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene‐editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells. The current review discusses feasible approaches to deliver the CRISPR/Cas9 systems, as well as potential strategies in gene‐editing for NK cell immunotherapy for cancers.

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Section: The Crispr/cas9 System and Its Deliverymentioning

confidence: 99%

Section: The Crispr/cas9 System and Its Deliverymentioning

confidence: 99%

Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9

Afolabi

Adeshakin

Sani³

et al. 2019

Immunology

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“…Recently, Cas9 RNP was modified with an asialoglycoprotein receptor and a NLS that can be recognized and internalized by liver cells. This biochemical‐modified RNP complex selectively gathers in the liver and conducts cell‐selective gene editing …”

Section: Delivery Of Crispr/cas9mentioning

confidence: 99%

“…This biochemical-modified RNP complex selectively gathers in the liver and conducts cell-selective gene editing. [46]…”

Section: Biochemical Modificationmentioning

confidence: 99%

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

et al. 2018

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Precise editing of the genome of a living body is a goal pursued by scientists in many fields. In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR‐associated) genome‐editing systems have become a revolutionary toolbox for gene editing across various species. However, the low transfection efficiency of the CRISPR/Cas9 system to mammalian cells in vitro and in vivo is a big obstacle hindering wide and deep application. In this review, recently developed delivery strategies for various CRISPR/Cas9 formulations and their applications in treating gene‐related diseases are briefly summarized. This review should inspire others to explore more efficient strategies for CRISPR system delivery and gene therapy.

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“…10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display.…”

mentioning

confidence: 99%

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

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Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC MAINCRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. 1 Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; 2,3 however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. For example, precise sequence alteration at the Cas9 cleavage site requires the efficient incorporation of exogenously supplied single-stranded oligonucleotide donor DNA (ssODN) 4 via the homology-directed repair (HDR) pathway. 5-7 However, most cells instead employ the nonhomologous end-joining (NHEJ) repair, which results in unpredictable insertions and deletions of bases at the cleavage site, some of which are large enough to have pathogenic consequences. 3,8,9 Displaying ssODNs on Cas9 can increase their local concentration around the DNA strand break site to allow enhanced incorporation of the desired sequence. In another application, appending PEG chains to Cas9 may reduce the immunogenicity of this bacterial protein. 10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display. Second, the platform should be robust and implementable by nonspecialists, given the diverse u...

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Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

Cited by 136 publications

References 48 publications

Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9

Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

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