CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip

Abstract

Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9-12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.

Keywords: CRISPR; Cas9 protein; genome; microfluidics; tissue engineering.

Figure 1

Combination of the CRISPR technology along with an electrochemical microfluidic biosensor for miRNA diagnostics. (a) Schematic of the off-chip miRNA targeting including the enzyme Cas13a, the target miRNA (blue), the target-specific crRNA, and the biotin and 6-FAM-labeled reporter RNA, which is immobilized after the cleavage process onto the streptavidin (SA) and BSA blocked channel surface. (b) Schematic of the single-stranded target miRNA, miR-19b, and the crRNA, where the complementary sequence is highlighted in blue. (c) Working principle and photo of the microfluidic biosensor with its main elements including the contact pads for the working, reference, and counter electrode (WE, RE, CE) in the electrochemical cell (marked in blue) and the immobilization area for the assay preparation (highlighted in red), separated by the hydrophobic stopping barrier SB, reproduced with permission from [36] Copyright 2020 Wiley.

Figure 2

Cas9 RNP delivery strategy and chip performance. (A) Experimental scheme of Cas9/crRNA/tracrRNA ribonucleoprotein (Cas9 RNP) delivery for genome editing in hard-to-transfect cells via a microfluidic cell deformation chip. Scanning electron microscopy of deformable zones in the device is also shown. Scale bar, 10 µm. The red arrow indicates one curved tunnel with a depth of 15 µm and a width of 4–8 µm. (B) Cell deformation was shown by microscopy when SK-BR-3 cells passed through the micro construction: scale bar, 10 µm. (C) Microscopy of SK-BR-3 cells into which FITC-labeled 70 kDa dextran molecules were delivered through different chip designs. Arrays 1 and 2 show cell passage curved tunnels formed by different structural arrangements. Scale bar, 20 µm. (D) Delivery efficiency and cell viability 16 h after treatment were calculated for (C) at a fluid speed of 150 µL min⁻¹. Array 1 × 10 or array 2 × 10 indicates cells passing through a chip with 10 repeats of identical cell deformation zones. Error bars indicate SEM (n = 3). (Reproduced with permission from [95] Copyright 2020 Wiley.