Figure 3.

Generation of the ΔTGM1 human cellular model

(A) TGM1 KO strategy design. gRNA1 and gRNA2 target E2, while gRNA3 targets E4 of the TGM1 gene. gRNA1 and gRNA2 differ in 1 nt (underlined), allowing targeting of both alleles, exploiting the presence of a SNP in a wild-type strain (K81). gRNA sequences are enclosed in colored boxes, with the protospacer adjacent motif (PAM) shown in bold. (B) PCR analysis on both edited exons performed on genomic DNA from wild-type keratinocytes (K81) and ΔTGM1-nucleofected keratinocytes. Note the intensity reduction of the bands at 608 bp and 780 bp in ΔTGM1 nucleofected keratinocytes. (C and D) RT-qPCR (C) and western blot analysis (D) show ablation of both TGM1 mRNA (C, orange bars) and protein (D) in ΔTGM1 keratinocytes as compared to wild-type cells. (E) Relative TG1 enzymatic activity in growing (3-day) and over-confluent (14-day) wild-type and ΔTGM1 keratinocytes. n = 3 replicates, data are presented as mean ± SD (^∗p < 0.0001).