Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

Abstract

CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.

Keywords: CRISPR; RNP; genome editing; nanoparticles; polymers.

Figure 1

Schematic illustration of the structure and molecular mechanism of the CRISPR/Cas9 system. A. structure of Cas9 protein and sgRNA. B. Formation of DSB via CRISPR/Cas9 system. C. The repair mechanisms of DSBs.

Figure 2

The physical approaches for Cas9 RNP delivery. A and B. Schematic diagrams of microinjection (A) and biolistics (B) for RNP delivery. C. Schematic of the NanoEP electroporation device. Reduced with permission from . Copyright 2019, National Academy of Sciences. D. Illustration of the original microfluidic device for macromolecules delivery via cell squeezing. Adapted with permission from . Copyright 2013, National Academy of Sciences. E. Workflow of the silicon microfluidic chip. F. Images showing the nanostructures of silicon nanotube. Reduced with permission from . Copyright 2020, Wiley-VCH. G. Schematic of the iTOP system.

Figure 3

Virus-like particles for Cas9 RNP delivery. A. Schematic of 'all in one virus' production. Adapted with permission from . Copyright 2016, Springer Nature. Creative Commons CC BY. B. Scheme describing the production of MLV-like particles. Reduced with permission form . Copyright 2019, Springer Nature. Creative Commons CC BY. C. Illustration of a lentivirus-like RNP delivery system. Adapted with permission from . Copyright 2019, Oxford University Press. Creative Commons CC BY.

Figure 4

Cell-derived extracellular vesicles for Cas9 RNP delivery. A. Packing strategy of recruiting Cas9 into ARMMs via specific interaction between WW domain and PPXY motifs of ARRDC1. Reprinted with permission from . Copyright 2018, Springer Nature. Creative Commons CC BY. B. Schematic of the production of RNP-packaging fusogenic VSV-G vesicles. Reprinted with permission from . Copyright 2018, Elsevier. Creative Commons CC BY-NC-ND. C. Selective packaging of Cas9 and sgRNA into extracellular nanovesicles. Adapted with permission from . Copyright 2020, Copyright Springer Nature. Creative Commons CC BY.

Figure 5

Intracellular delivery of Cas9 RNP by lipids. A. Cationic lipid-mediated delivery of CRISPR system by RNP complex or fusing Cas9 protein with anionic GFP. B. Bioreducible cationic lipid library for the delivery of genome editing systems . C. Expansion of bioreducible cationic lipid library for Cas9 RNP delivery . D. Synthesis of cationic chalcogen-containing lipids for Cas9 RNP delivery . E. Non-cationic NTA-containing lipidoids for Cas9 RNP delivery. Red color identifying the leading amine heads or lipidoid for the intracellular delivery of Cas9 RNP .

Figure 6

Lipid vehicles for Cas9 RNP delivery. A. Lecithin-based liposomal delivery system for Cas9 RNP delivery. Reduced with permission from . Copyright 2019, Springer Nature. Creative Commons CC BY. B. Illustration of T-shape lipo-OAAs with different fatty acids, in which lipo-OAA-containing OHSteA was superior to others in higher genome editing efficiency. Reduced with permission form . Copyright 2020, American Chemical Society. C. A fluorescent surfactant used to enhance the Cas9 RNP delivery of lipofectamine. Adapted with permission from . Copyright 2019, Royal Society of Chemistry.

Figure 7

CPP- and lipopeptide-based delivery systems. A. CPP-conjugated Cas9 protein and CPP complexed sgRNA for intracellular delivery. Reduced with permission from . Copyright 2014, Cold Spring Harbor Laboratory Press. Creative Commons CC BY. B. Schematic of chimeric Cas9-LWMP complexed with dual RNAs. Reduced with permission from . Copyright 2018, American Chemical Society. C. Amphipathic α-helical peptides for the intracellular delivery of Cas9 RNP without covalent conjugation. Reduced with permission from . Copyright 2018, American Society for Biochemistry and Molecular Biology. Creative Commons CC BY. D. Illustration of the lipopeptide formed via a supramolecular strategy for the screening of Cas9 RNP delivery. Adapted with permission from . Copyright 2017, Royal Society of Chemistry. Creative Commons CC BY-NC.

Figure 8

Polymers for Cas9 RNP delivery. A. PBA-rich dendrimer used for the intracellular delivery of protein and Cas9 RNP. Adapt with permission from . Copyright 2019, The Authors, some rights reserved. Creative Commons CC BY-NC. B. Carboxylated branched PBAEs used for the intracellular delivery of protein and Cas9 RNP. Reprinted with permission from . Copyright 2019, The Authors, some right reserved. Creative Commons CC BY. C. Illustration of the assembly of pH-responsive PEGylated PLL and double targeted Cas9 RNPs. Reduced with permission from . Copyright 2019, American Chemical Society.

Figure 9

Nanogels for the intracellular delivery of Cas9 RNP. A. Schematic illustration of the RNP-embedded nucleic acid nanogel formation and intracellular delivery. Reduced with permission from . Copyright 2019, Royal Society of Chemistry. B. Image of design and preparation of reduction-responsive nanogel for Cas9 RNP delivery.

Figure 10

GNP-based delivery platforms for Cas9 RNP. A. Rational design of arginine-functionalized GNPs for the intracellular delivery of E-tagged Cas9 or RNP. Adapted with permission from . Copyright 2017, American Chemical Society. B. Schematic illustration of pH-induced assembly of GSH-modified GNPs with Cas9 protein. Reduced with permission from . Copyright 2019, American Chemical Society. C. PAsp(DET) coated SNAs for the delivery of Cas9 RNP. D. Schematic illustration of GNP-based RNP nanoformulation for genome editing.

Figure 11

Inorganic materials for the intracellular delivery of Cas9 RNP. A. Illustration of the encapsulation of Cas9 RNP into ZIF-8. Reprinted with permission from . Copyright 2017, American Chemical Society. B. Schematic illustration of the self-assembly and ATP-triggered release of ZIF-90/RNP complex. Reprinted with permission from . Copyright 2019, American Chemical Society. C. Schematic diagram of the GO-PEG-PEI based Cas9 RNP delivery system. Adapted with permission from . Copyright 2018, Royal Society of Chemistry. D. Image of the complexation of BP nanosheets and Cas9-3NLS RNPs for genome editing. Adapted with permission from . Copyright 2018, Wiley-VCH.

Figure 12

DNA nanoclews for the delivery of Cas9 RNP. Adapted with permission from . Copyright 2015, Wily-VCH.

Figure 13

Responsive delivery systems for RNP delivery. A. NPOM-caged sgRNA for spatiotemporal control of Cas9 RNP function. Adapted with permission from . Copyright 2020, Wiley-VCH. B. UCNP-based NIR-responsive Cas9 RNP delivery system. Reduced with permission from . Copyright 2019, The Authors, some rights reserved. Creative Commons CC BY-NC. C. Schematic illustration of US-activatable microbubbles as Cas9 RNP delivery system for androgenic alopecia therapy.

Figure 14

Reduction-sensitive Cas9 RNP delivery systems. A. Synthesis of GSH-responsive cationic block copolymer for the delivery of CRISPR/Cas9 system. Reduced with permission from . Copyright 2018, American Chemical Society. B. Redox-responsive cross-linked polymers for the delivery of Cas9 RNP. Adapted with permission from . Copyright 2018, American Chemical Society. C. Schematic illustration on microenvironment-responsive delivery of Cas9 RNP. Reduced with permission from . Copyright 2019, American Chemical Society.

Figure 15

Gal-mediated targeted Cas9 RNP delivery. A. Receptor-mediated delivery of Cas9 RNP. Reduced with permission from . Copyright 2018, American Chemical Society. B. Schematic diagram of Gal-conjugated gold nanoclusters for Cas9 RNP delivery. Reduced with permission from . Copyright 2019, Wiley. C. Schematic illustration of the Gal-targeted PEI nanoparticles for genome editing. Adapted with permission from . Copyright 2020, The Authors, some rights reserved. Creative Commons CC BY-NC.

Figure 16

Targeted delivery systems for Cas9 RNP delivery. A. iRGD-containing lipopeptide for targeted Cas9 RNP delivery. Reduced with permission from . B. Folate-based targeted delivery system for Cas9 RNP delivery. Reduced with permission from . Copyright 2019, Wiley-VCH. Copyright 2019, Royal Society of Chemistry. Creative Commons BY-NC. C. Schematic illustration of the cell-specific delivery system. Reduced with permission from . Copyright 2020, American Chemical Society.

Figure 17

Selective organ targeting systems for the delivery of CRISPR/Cas9 system .