Cas9 Ribonucleoprotein Delivery via Microfluidic Cell‐Deformation Chip for Human T‐Cell Genome Editing and Immunotherapy

Abstract

This study reports a microfluidic cell deformation‐based method to deliver the Cas9 ribonucleoprotein (RNP) complexes to different cell types for efficient genome editing, including hard‐to‐transfect human primary CD4+ T cells. The RNP based CRISPR‐Cas9 system has great advantage in shortening reaction time and reducing off‐target problems, which holds great potential in future gene therapy applications.

Biochips—New Platforms for Cell‐Based Immunological Assays

“…High delivery efficiency of different macromolecules into different cell types as well as high cell viability have been demonstrated, including delivery to hard‐to‐transfect lymphoma cells and embryonic stem cells . We also demonstrated that Cas9 ribonucleoprotein delivery using our cell deformation device achieved highly efficient genome editing and reduced off‐target effects for primary T cells, which are known as hard to transfect (Figure c) . Furthermore, knock‐in of programmed cell death protein 1 (PD‐1/PDCD‐1), a validated target for tumor immunotherapy, to the genomes of primary T cells was achieved.…”

Viral Generation, Packaging, and Transduction on a Digital Microfluidic Platform

“…Since genome editing efficiency varies with different cell lines, we implemented the gene editing workflow on H1299 cells and similarly observed the successful knockout of integrated eGFP (gene cleavage efficiency of 16.1%) (Figure S10). Although downstream gene editing analysis of CRISPR knockouts from microfluidic devices has been shown previously, − this is the first demonstration showing the integration of lentiviral packaging, generation, and transduction on a microfluidic device followed by downstream gene editing analysis (single clone isolation and expansion).…”

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

“…In addition, knock-in genome amendment in primary T cells was achieved in PD-1 as a programmed cell death protein because creating the genetic deletion in PD-1 was proven advantageous in engineering T cells for cancer and tumor immunotherapies. According to this study, microfluidic cell deformation-based Cas9 RNP delivery provided accurate genome editing in T cells and cancer immunotherapy [95].…”