Identification of Proteins Involved in Cell Membrane Permeabilization by Nanosecond Electric Pulses (nsEP)

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R > 0.9, p < 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP.

Automated summary

The study aimed to identify endogenous proteins that either facilitate or impede the permeabilized state of the cell membrane disrupted by nsEP. Knockouts (KOs) of 316 genes encoding for membrane proteins were generated using a LentiArray CRISPR library in U937 human monocytes expressing Cas9 nuclease. Only two KOs for SCNN1A and CLCA1 genes showed a significant reduction in Yo-Pro-1 uptake, indicating their potential involvement in electropermeabilization lesions or lifespan. However, as many as 39 genes were identified as likely contributors to membrane stability or repair after nsEP.