Additional file 2: of Genome editing through large insertion leads to the skipping of targeted exon

Figure S2. Strategy for Cas9-mediated generation of hCDC14A knockout RPE1 cells. (A) Workflow for the construction of RPE1 hCDC14A-KO cell line. pCW-Cas9 plasmid containing doxycycline (Dox) inducible spCas9 was lentivirally integrated into RPE1 FRT/T-Rex cells. (B, C) Successful expression and nuclear localization of Cas9 was confirmed by indirect-immunofluorescence (B) and western blotting (C). (D) Junction PCR with forward primer in NeoR cassette and reverse primer in the genome outside homology arm (as in Fig. 1a) confirmed successful targeting and insertion of the selection marker. Exon skipping was confirmed by RT-PCR (primers as in Fig. 3a). Presence of both wild type and exon-skipped RNA indicated the targeting of single allele in clones 1 and 20. (PDF 1952 kb)