Supplementary Figure 2 from Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine

Generation of CT26 #5KO and CT26 #23KO cells using CRISPR-Cas9 technology. A) CRISPR/Cas9 strategy used for the generation of CT26 cells lacking of nAg #5. Red arrows represent forward and reverse primers used for PCR. Blue lines 1 and 2 indicate 2 sgRNAs designed for Cas9-mediating genome editing, within exon 10, harboring #5 mutation (blue star). The position of the 2 sgRNAs is also indicated. B) Left panel: representative agarose gel of PCR screening on cDNA showing homozygous deletions of 74nt in the genomic region encoding nAg#5 (E2f8 gene). Right panel: representative sequenced region of E2f8 gene and the relative chromatogram. In red, PAM sequences are indicated. The deleted region is underlined. The mutated nucleotide of nAg#5 is in black bold. Red arrow indicates Cas9 cut site. C) CRISPR/Cas9 strategy used for the generation of CT26 cells lacking of nAg #23. Red arrows represent forward and reverse primers used for PCR. Blue lines indicate the 2 sgRNAs designed for Cas9-mediating genome editing. Exon 12 harbors #23 mutation (green star). The position of the 2 sgRNAs is also indicated. D) Left panel: representative agarose gel of PCR screening on genomic DNA showing homozygous deletions of 116nt in the genomic region encoding nAg#23 (Mtch1 gene). Right panel: representative sequenced region of Mtch1 gene and relative chromatogram. In red, PAM sequences are indicated. The deleted region is underlined. The mutated nucleotide of nAg#23 is in black bold. Red arrow indicates Cas9 cut site.