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.
摘要：
CRISPR基因组编辑是一种广泛用于干扰细胞和组织内感兴趣基因的工具，可以用作研究基因型和细胞表型之间联系的研究工具。由于低转染效率或单细胞生存能力，高效基因组编辑在某些细胞类型中受到限制。这对BeWo细胞来说是正确的,胎盘合胞体滋养层细胞-细胞融合和激素分泌的体外模型。在这里，我们描述了一种优化且易于使用的方案，用于使用通过电穿孔递送的CRISPRCas9核糖核蛋白（RNP）复合物在BeWo细胞中敲除。Further,我们描述了成功的指导RNA设计的参数，以及如何评估BeWo细胞中的基因敲除，以便用户可以将该技术应用于自己感兴趣的基因。我们为诱导细胞-细胞融合蛋白Syncytin-2（ERVFRD-1）的高效敲除和评估该基因座的编辑效率提供了阳性对照。我们预计，BeWo细胞中有效的RNP介导的基因敲除将有助于在这个重要的细胞模型系统中研究参与细胞-细胞融合和激素分泌的新基因。此外，这种优化的核转染和RNP递送策略可用于其他难以编辑的滋养层细胞,或者可用于将转基因有效递送至BeWo细胞.
