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. 2020 Aug 22;12(9):2378. doi: 10.3390/cancers12092378

Figure 1.

Figure 1

Human CD44 locus and CRISPR/Cas9 single guide RNA (sgRNA) design strategy. (A) The human CD44 canonical transcripts annotated with 18 exons (ENST00000428726.8). The dark grey exons comprise the constitutively expressed portion of the gene (exons 1–5, 16–18 and 20), and the light grey corresponds to the alternatively expressed portion of the gene in humans (exons v2–v10). Each exon is docked to adjacent exons in the mRNA sequence. Every set of three nucleotides encodes the amino acids that will comprise the CD44 protein. In the exon boundaries, the resultant amino acid may enclose the last nucleotide of the former exon and the two first nucleotides from the next exon (>), or contain the two last nucleotides of the former exon and the first nucleotide from the next exon (<). This indicates that if exon-v6 is removed, exons v5 and v7 are spliced in-frame. (B) Genomic spots of the sgRNA designed to target the adjacent areas to exon-v6. Primers B and E (represented in grey) were used to genotype the complementary DNA (cDNA) and primer C and F (represented in black) were used to genotype the genomic DNA (gDNA) of the edited cells. (C) Specifications of the sgRNAs (g1–g6) designed near the exon-v6 vicinity to permanently induce exon-v6 skipping. TM—Transmembrane domain; CP—Cytoplasmic domain; WT—wild-type; PAM—Protospacer Adjacent Motif.