{Reference Type}: Journal Article
{Title}: Optimized CRISPR-based knockout in BeWo cells.
{Author}: Yin E;Esbin MN;
{Journal}: Placenta
{Volume}: 0
{Issue}: 0
{Year}: 2024 Jul 8
{Factor}: 3.287
{DOI}: 10.1016/j.placenta.2024.07.005
{Abstract}: CRISPR genome editing is a widely used tool to perturb genes of interest within cells and tissues and can be used as a research tool to study the connection between genotypes and cellular phenotypes. Highly efficient genome editing is limited in certain cell types due to low transfection efficiency or single-cell survivability. This is true for BeWo cells, an in vitro model of placental syncytiotrophoblast cell-cell fusion and hormone secretion. Here we describe an optimized and easy-to-use protocol for knockout in BeWo cells using CRISPR Cas9 ribonucleoprotein (RNP) complexes delivered via electroporation. Further, we describe parameters for successful guide RNA design and how to assess genetic knockouts in BeWo cells so that users can apply this technique to their own genes of interest. We provide a positive control for inducing highly efficient knockout of the cell-cell fusion protein Syncytin-2 (ERVFRD-1) and assessing editing efficiency at this locus. We anticipate that efficient RNP-mediated genetic knockouts in BeWo cells will facilitate the study of new genes involved in cell-cell fusion and hormone secretion in this important cellular model system. Furthermore, this strategy of optimized nucleofection and RNP delivery may be of use in other difficult-to-edit trophoblast cells or could be applied to efficiently deliver transgenes to BeWo cells.