Siraj, N. | bioRxiv Science

bioRxiv2025

Generation of Cellular Biofactories for Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing

Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods using transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we developed CRISPR-Cas9 engineered HEK293T cell lines with stable integration of mCherry-C1C2 fusion proteins at the AAVS1 locus for continuous production of surface-modified EVs. The engineered cell lines demonstrated significantly higher surface display efficiency compared to transient transfection, with reduced batch-to-batch variability. EVs maintained native characteristics including size distribution (120-130 nm) and marker expression while showing efficient cellular uptake. The platform maintained consistent production of uniformly modified EVs with stable transgene expression over at least 25 passages (~3 months), eliminating the need for repeated transfections and reducing batch-to-batch variability inherent to transient expression systems.

bioengineering↗

bioRxiv2025

Understanding Structural Mechanics of Ligated DNA Crystals via Molecular Dynamics Simulation

DNA self-assembly is a highly programmable method that can construct arbitrary architectures based on sequence complementarity. Among various constructs, DNA crystals are macroscopic crystalline materials formed by assembling motifs via sticky end association. Due to their high structural integrity and size ranging from tens to hundreds of micrometers, DNA crystals offer unique opportunities to study structural properties and deformation behaviors of DNA assemblies. For example, enzymatic ligation of sticky ends can selectively seal nicks resulting in more robust structures with enhanced mechanical properties. However, the research efforts have been mostly on experiments such as different motif designs, structural optimization, or new synthesis methods, while their mechanics are not fully understood. The complex properties of DNA crystals are difficult to study via experiment alone, and numerical simulation can complement and inform the experiment. Coarse-grained molecular dynamics (MD) simulation is a powerful tool that can probe the mechanics of DNA assemblies. Here, we investigate DNA crystals made of four different motif lengths with various ligation patterns (full ligation, major directions, connectors, and in-plane) using oxDNA, an open-source, coarse-grained MD platform. We find that several distinct deformation stages emerge in response to mechanical loading and that the number and the location of the ligated nucleotides can significantly modulate structural behaviors. These findings should be useful for predicting crystal properties and thus improving the design.

biophysics↗