Abstract

Simple SummaryCRISPR/Cas9 system as a potential gene editing platform has been widely applied in biological engineering and disease therapies. To achieve precise gene targeting, active CRISPR/Cas9 components must be efficiently transported to targeted cells. As a simple and effective strategy, Cold Atmospheric Plasma (CAP) treatment has been demonstrated for the transmembrane delivery of various exogenous materials. In comparison with carrier-dependent delivery methods, this carrier-free platform provides a promising alternative to circumvent the obstacles of biosafety and complicated preparation processes. In this work, a CAP-based CRISPR/Cas9 carrier-free delivery platform has been established and corresponding mechanism related to efficient transportation has been explored. Briefly, the efficient production of bioactive species in culture media after CAP treatment alters cell membrane potential and permeability, which facilitates cytosolic delivery of active CRISPR/Cas9 components via passive diffusion and ATP-dependent endocytosis pathways, resulting in efficient genome editing and gene silencing. This carrier-free strategy using CAP-based transportation may also be extended to other active biomolecules in drug delivery and gene therapy.AbstractA carrier-free CRISPR/Cas9 ribonucleoprotein delivery strategy for genome editing mediated by a cold atmospheric plasma (CAP) is described. The CAP is promising in many biomedical applications due to efficient production of bioactive ionized species. The MCF-7 cancer cells after CAP exposure exhibit increased extracellular reactive oxygen and nitrogen species (RONS) and altered membrane potential and permeability. Hence, transmembrane transport of Ca2+into the cells increases and accelerates ATP hydrolysis, resulting in enhanced ATP-dependent endocytosis. Afterwards, the increased Ca2+and ATP contents promote the release of