CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

Figure 3

Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch < 4). Similarities are presented as dots, and mismatch sites are indicated by nucleotide substitution. (h) Sanger sequencing of SW579 cells infected with SC and EGFR sgRNA_2 virus was used to examine potential indels in off-target candidate genes.

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