Fig. 2

The E1 and E1.2 segments regulate MMP9 gene and protein expression. (a) Outline of the CRISPR-Cas9 mediated knockout method using Ribonucleoprotein complexes (RNPs). Two guide RNAs (gRNA) were designed, and each gRNA was fluorescently labeled with either MFP488 or Cy5 and fused with the Cas9 protein to assemble the CRISPR/Cas9-gRNA RNP complex. This complex was then reverse-transfected into the HTR-8/SVneo cell line. Dual-labeled cells were sorted and screened to confirm the knockout of E1 and E1.2. (b) The location of the gRNAs used to knockout the E1 segment (E1-KO). (c) The E1 knockout cell lines (E1-KO_c1 and E1_KO_c2) have significantly decreased MMP9 gene expression compared to the wild-type (WT) cell line. (d) Images and (e) quantification of western blots showing that MMP9 protein expression was significantly decreased in the E1-KO_c1 and E1-KO_c2 cell line compared to the WT cell line. (f) The location of the gRNAs used to knockout the E1.2 segment (E1.2-KO). (g) The E1.2 (E1.2-KO_c1 and E1.2-KO_c2) knockout cell lines have significantly increased MMP9 gene expression compared to the WT cell line. (h) Images and (i) quantification of western blots showing that MMP9 protein expression was significantly increased in the E1.2-KO_c1 and E1.2-KO_c2 cell lines compared to the WT cell line. In (b) and (f), the black bar represents the E1 and E1.2 segments respectively, and the inverted triangles are the nucleotide locations where the gRNA was designed to cut. The colors of the triangles correspond to the different fluorescent labels, MFP488 (green) or Cy5 (red). The black dots in (c), (e), (g) and (i) are individual biological replicate values for the experiment. Details of antibody concentration and cell counts used for western blots are provided in Supplementary Table S9. (a), (b) and (f) were created with BioRender.com. (**) indicates p-value < 0.01, and (***) indicates p-value < 0.001. All experiments were performed with three biological replicates. Results are displayed as the mean +/- standard error (SE) and p-values were calculated using the two-tailed student’s t-test.