CRISPR/Cas9-mediated gene editing in trophoblast cells via mechanoporation for preeclampsia insight

Preeclampsia is a severe pregnancy complication marked by impaired trophoblast function and abnormal placental development, leading to significant maternal and fetal morbidity. FK506-binding protein-like (FKBPL) has been identified as a potential biomarker as it is significantly downregulated in early pregnancy stages of women who progress to develop preeclampsia. However, editing the Fkbpl gene in trophoblast cells to create a model of preeclampsia using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is challenging due to inefficient delivery, leading to low editing efficiency and reduced cell viability. To address these challenges, we developed a cost-effective and minimally invasive mechanoporation system using micro-engineered filters to deliver CRISPR/Cas9 plasmid DNA (pDNA) targeting the Fkbpl gene into trophoblast cells. This approach successfully generated cell lines with a 38% knockout (K/O) of Fkbpl expression, significantly reducing cell migration (wildtype (WT): 28.77% ± 4.7 vs. 38% K/O: 4.95% ± 0.8, wound closure, **p < 0.01) and proliferation (WT: 1.26 ± 0.06 vs. 38% K/O: 0.81 ± 0.01, ****p < 0.0001). Lower Fkbpl-K/O efficiency of 17% showed a similar reduction in cell proliferation as the 38% K/O clone. Although a full Fkbpl-K/O in the ACH-3P first-trimester trophoblast cell line was not achieved, the partial K/O provided valuable insights into Fkbpl's role in trophoblast function relevant to preeclampsia pathogenesis. Moreover, treatment with mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) or MSC-sEVs did not restore migratory capacity in Fkbpl-deficient cells (p = 0.14). MSC-sEVs increased proliferation in WT ACH-3P cells at 1 µg (p < 0.05) and 2 µg (p < 0.01) doses, however, were not effective in either 17% or 38% Fkbpl-K/O clones, suggesting that FKBPL is an important mechanism of MSC-sEV-mediated therapeutic effect in trophoblasts in the context of preeclampsia. This study advances gene-editing techniques in placental biology and proposes new therapeutic strategies and mechanisms for pregnancy-related complications. A Schematic overview of CRISPR/Cas9 plasmid delivery using microfiltroporation compared to gold standard electroporation and lipofection technologies in trophoblast cells. A CRISPR/Cas9 plasmid targeting Fkbpl was delivered to the first trimester trophoblast cell line, ACH-3P. Cells were sorted according to green fluorescence protein (GFP) expression, expanded and assessed for changes in cell function using proliferation and migration assays. B Actual images of the isopore silicon nitride (SiN) microfilters used in this study and diagram of cell membrane dynamics in response to mechanoporation. This figure was created with Biorender.com. CRISPR clustered regularly interspaced short palindromic repeats, EP electroporation, MFP microfiltroporation.