Generation of hTfR1 knock-out cells using CRISPR/Cas9. (A) TFR1 locus on the human chromosome 3 targeted by CRISPR/Cas9. A sgRNA consisting of 20-nt guide sequence (blue) and a scaffold (green). The guide sequence anneals with the DNA target (blue bar) upstream of PAM (red). A red arrow points to the expected cleavage site. (B) Oligonucleotide sequences used for the generation and characterisation of Hs578TΔhTfR1 cells. (C) Detection of mutations in Hs578T clones generated by transduction of lentiCRISPR vector containing sgRNA targeting hTFR1and Cas9 nuclease. PCR products (1100 bp) containing the target site were denatured, re-annealed and probed with T7 endonuclease that recognizes non-perfectly matched DNA. A cleavage product of ~600 bp indicates successful NHEJ-mediated mutation in at least one copy of hTFR1 (lanes 2–11). Analysis of 12 clones is shown; (the amount of PCR product loaded in lane 4 was lower than in other lanes). (D) Western blot analysis of the hTFR1 knock-out Hs578T clone (−/−) together with controls including parental cells (WT) and a clone containing at least one copy of non-mutated hTFR1(+/+). Cellular lysates were also probed for actin to ensure equal loading (bottom panel). (E) Sequence analysis of the hTFR1 target site of the clone bearing mutations in all three copies of the target site in Hs578T cells.