A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.

Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA.

CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.

Localized delivery of coding nucleic acids into adherent cells by in situ electroporation: integrated impedance-based monitoring allows for loss-of-function or gain-of-function screening

Coding nucleic acids (NAs) like DNA, mRNA or siRNA hold endless potential to engineer cells with respect to their genome and proteome. However, minimally invasive delivery of NAs across an intact plasma membrane into the cytosol of living cells remains a technical challenge. Electroporation creates transient pores in the plasma membrane by applying well-defined electric fields for fractions of a second allowing NA transfer across the membrane. In situ electroporation (ISE) applies the electric field to adherent cells growing on the surface of planar, thin-film electrodes deposited on the bottom of the culture well. This study reports on the localized delivery of plasmid DNA, mRNA, siRNA and aptamers into different mammalian cell lines by ISE. Fluorescence microscopy verified successful delivery of labeled NAs themselves (siRNA, aptamers) or expression of the proteins they encoded (DNA, mRNA). Impedance readings of the cell-covered electrodes before and after ISE allowed tracking the invasiveness of the pulse, the recovery of the cells within 30 min and the phenotypic impact of the NAs inside the cells. Loading the cells with siRNA specifically designed to knock down gene transcripts essential for survival, led to the onset of cell death within 20 h after the pulse. The combination of ISE, specific NAs and impedance-based cell monitoring paves the way for a new class of gain-of-function or loss-of-function experiments.

A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos

A lipid nanoparticle platform for high yield CRISPR-targeted homology directed repair enables fully non-viral CAR T cell generation

Optimization of CRISPR dCas9 Lentiviral Transduction in Nucleus Pulposus Cells for Use in Tissue Engineering.

An optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.

Highly efficient CRISPR editing enabled by magnetic nanoparticle delivery

Efficient delivery of CRISPR components remains a major determinant of genome editing outcomes. In this study, we compared conventional lipofection with magnetic nanoparticle-assisted gene delivery (magnetofection) for CRISPR-mediated genome editing efficiency using SpCas9 and AsCas12a systems. Based on the average values obtained from multiple independent targets, lipofection resulted in relatively low indel efficiencies, with mean values of average 8.1%–12.47%. In contrast, magnetofection markedly enhanced genome editing outcomes, yielding average indel efficiencies of average 42.29%–45.04%, representing a substantial increase (3.39- and 5.56-fold, respectively) compared with lipofection. This enhancement was consistently observed across both SpCas9-and AsCas12a-mediated editing, indicating that the improved efficiency conferred by magnetic nanoparticle delivery is independent of the nuclease platform. Furthermore, the increased performance of magnetofection was reproducible across multiple genomic loci and cell lines and was also effective under RNP-based delivery conditions, demonstrating its robustness and reliability. In addition to indel-based genome disruption, magnetofection also significantly improved prime editing efficiency (13.95% on average) compared to lipofection (3.81% on average). Overall, our results demonstrate that magnetic nanoparticle-mediated delivery enables highly efficient and reproducible CRISPR genome editing, substantially outperforming conventional lipofection for both indel formation and prime editing. Magnetofection therefore represents a powerful and broadly applicable delivery strategy for next-generation genome editing applications.