Functional characterization of SLC26A3 c.392C>G (p.P131R) mutation in intestinal barrier function using CRISPR/CAS9-created cell models

Construction and expression of P131R-SLC26A3 genetic variant on Caco-2 cells. a The SNP rs386833481 in the coding sequence of the SLC26A3 gene leads to the Proline to Arginine amino acid change at position 131. b Topographic model of hSLC26A3 (reproduced from Wedenoja et al. [3]) showing the predicted location of P131R within the transmembrane domain. c Alignment of mammalian SLC26A3 polypeptide sequences in the region of hSLC26A3 P131R (Highlight), showing completely conservation among species orthologs (CLUSTAL 2.1-multiple sequence alignment). d Schematic of the RNA-guided Cas9 nuclease. The Cas9 nuclease from S. pyogenes (in yellow) is targeted to human SLC26A3 P131R locus by a sgRNA consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence pairs with the DNA target (blue bar on top strand), directly upstream of a requisite 5′-NGG adjacent motif (PAM; pink). Cas9 mediates a DSB？~？3 bp upstream of the PAM (red triangle). e Results of TaqMan？ Genotyping Assay of DNA isolated from either wild-type Caco-2 cells (CC) or CRIPSR/Cas9 gene edited cells: (CG) and (GG). CRISPR/Cas9 transfected Caco-2 cells were selected with puromycin. Three CRISPR/Cas9n-Caco-2 puromycin resistant lines were tested. N？=？3 TaqMan Assays per sample. f Results of Sanger sequencing for confirming the construction of P131R-SLC26A3 on Caco-2 cells by CRISPR/Cas