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. 2023 Jul 20;13(14):e4724. doi: 10.21769/BioProtoc.4724

Figure 3. Induction of +1 frameshift and premature translational stop by homology-directed repair (HDR) of ssDNA template.

Figure 3.

(A) Overview of exon 3 (dark blue) and 414 bp amplicon (yellow) spanning the homology repair-directed template insert (HDR template HindIII +1fs, dark green, homology arms depicted in light green) carrying a HindIII restriction site (orange). Alternative stop codon [stop (ORF +2)] on position 138–141 of wildtype PD-L1 exon 3 is depicted in red. Primers spanning the amplicon are depicted in purple. (B) Sequence of 111 bp HDR template Hind III +1fs as depicted in CLC Genomics Workbench. 57 bp (left) and 50 bp (right) homology arms span the 4 bp (AGCT) HindIII restriction site insert. Insertion of 4 bp HindIII restriction site replacing 3 bp (CGA) of wildtype PD-L1 leads to a +1 frameshift and a premature stop of translation due to the alternative stop codon in ORF +2.