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

Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells

Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.

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.

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

Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4⁺ and CD8⁺ T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.

A PepFect14 analog improves non‐viral CRISPR delivery in primary human cells to facilitate genome editing and repair

A PepFect14 analog is employed to deliver high-fidelity Cas9-ribonucleoproteins and non-viral repair templates into primary human skin cells to mediate gene editing and repair targeting genes underlying the group of genetic skin blistering disorders epidermolysis bullosa (EB).

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

Recent advances of CRISPR-based gene editing technologies and delivery strategies.

The types, principles and characteristics of gene editing systems are introduced in order to understand their requirements for delivery tools and to provide new insights to facilitate appropriate delivery systems or improve the efficacy of existing systems.