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

Figure 2

EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

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