Fig. 4

Strategy for Cas9-mediated generation of hCDC14A knockout HCT116 cells. a, b Workflow for CRISPR-Cas9 medicated insertion of NeoR into exon 9 of hCDC14A. Guide RNAs (gRNAs) targeting the exon 9 of hCDC14A gene were designed using the web tool (http://crispr.mit.edu/ [25]). ‘Churh gRNA insert’ containing the U6 promoter and gRNA scaffold [26] was first synthesized as gBlock and cloned into pJet. The intended gRNAs were inserted through PCR mutagenesis using primers indicated by arrows (top of b). The same donor construct (bottom of b) as in case of ZFN-mediated genome editing was used to target the locus. c Junction PCR (as in Fig. 1a) with forward primer in NeoR cassette and reverse primer in the genome outside homology arms confirmed successful targeting and insertion of the selection marker.d RT-PCR of purified mRNA from Wt and different CRISPR-Cas9 targeted hCDC14A-KO clonal cells. Primers were as in Fig. 3a. Presence of both wild type and exon-skipped RNA indicated the targeting of single allele in clone 17. The other NeoR clones 7, 15 and 19 contained bi-allelic knockout of the hCDC14A gene. e The generated DNA bands of the RT-PCR were gel purified and sequenced. Sequences of alternating exon junctions of hCDC14A are shown and in-frame exon skipping can be deduced from the amino acid sequences written above the codons