A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing

Abstract

Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods.

Keywords: CRISPR delivery; gene editing; intracellular delivery; micro/nanotechnology; physical transfection; transfection methods.

Figure 1

Physical forces responsible for CRISPR transfection. The CRISPR/Cas9 system can be delivered as plasmid DNA, mRNA or RNP. The driving forces for CRISPR delivery include external field such as electrical, acoustic, laser/thermal and magnetic forces. Direct physical contact such as microinjection and passing constriction can also mediate CRISPR delivery.

Figure 2

Mechanical transfection platform deforms cell membrane. A. Cell squeezing device. Reproduced with permission from Proceedings of the National Academy of Sciences USA Copyright 2013. B. Workflow schematic and SEM image of sharp angle constriction made of silicon, scale bar: 30 µm. Reproduced with permission from Oxford University Press Copyright 2017. C. Image of cells being deformed when passing through the constriction. Reproduced with permission from American Association for the Advancement of Science CC BY-NC 4.0. D. Setup of TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF) method for CRISPR transfection. Reproduced with permission from Springer Nature CC BY 4.0.

Figure 3

Novel electroporation platform for transfection. A. Schematic of 3D microfluidic electroporation system. Reproduced from Ref. with permission from The Royal Society of Chemistry. B. Design for in situ electroporation microsystem. Reproduced with permission from Springer Nature CC BY 4.0. C. Cuvette design for tube electroporation. Reproduced with permission from Springer Nature CC BY 4.0. D. Nanostructure electroporation system (left) and corresponding mRNA transfection efficiency showing higher uniformity (red) compared to lipofection (gray) (right). Reproduced with permission from American Association for the Advancement of Science CC BY-NC 4.0.

Figure 4

Acoustoporation methods for cell transfection.

Figure 5

Laser optoporation methods for cell transfection. A. Workflow of transfection principles using laser optoporation. Reprinted from , Copyright 2013, with permission from Elsevier. B. On demand transfection triggered via laser and responsive particle. Reproduced with permission from Copyright 2019 Wiley.