# Membrane repair following filtroporation-induced cell permeabilization

## TL;DR

Filtroporation is a method to deliver gene-editing tools into cells with minimal disruption and faster membrane repair compared to other techniques.

## Contribution

Filtroporation causes fewer transcriptional changes than electroporation and involves multiple membrane repair mechanisms.

## Key findings

- Filtroporation enables efficient beta globin gene editing in hematopoietic stem and progenitor cells.
- Membrane repair occurs within 30 seconds after filtroporation, with calcium signaling playing a key role.
- Three mechanisms involving GRAF1, SNAP23, and CHMP4B contribute to membrane repair following filtroporation.

## Abstract

Mechanical disruption of cell membranes to enable intracellular delivery has been gaining traction as a methodology. Here, we show that a mechanical disruption-based strategy, filtroporation (FP), can be applied to edit the beta globin gene efficiently in human hematopoietic stem and progenitor cells. Gene expression analyses from RNA-Seq datasets demonstrate that electroporation (EP) yields greater transcriptional changes compared to FP globally, and gene pathway enrichment analyses suggest EP promotes stem cell differentiation while FP promotes cell division. Membrane repair occurs within 30 s of disruption by FP, and calcium signaling plays a key role in membrane repair in this context. Studies with fluorescently tagged membrane repair proteins, GRAF1, SNAP23, and CHMP4B, implicate the involvement of three mechanisms to reconstitute the cell barrier following FP. This work supports the evidence that the choice of intracellular delivery method affects transcriptional stem cell profile, and that membrane repair after mechanical disruption is a fast, multi-pathway process.

•Filtroporation enables delivery of CRISPR/Cas9 reagents and editing at the beta globin locus•Filtroporation causes fewer transcriptional changes than electroporation•Cells repair their membranes within 30 s of filtroporation•Multiple membrane repair mechanisms are involved following filtroporation

Filtroporation enables delivery of CRISPR/Cas9 reagents and editing at the beta globin locus

Filtroporation causes fewer transcriptional changes than electroporation

Cells repair their membranes within 30 s of filtroporation

Multiple membrane repair mechanisms are involved following filtroporation

Cell biology; Omics