Figure 2
Construction of TALENs-expressing plasmids applied in the human Nrf1 gene editing.
(a) Schematic diagram of TALENs-mediated editing of the human genomic Nrf1 sequence. A pair of TALEN-left and TALEN-right [either comprises nuclear localization signal (NLS), DNA-binding domain (DBD), activation domain (AD) and the fusion nuclease FOK1] were designed to recognize the boxed target sequences in close proximity to the site responsible for the translation start codon. (b) An assembly of the repeat modular DBD-coding cDNA sequences was made according to the ‘protein-DNA’ code as a guiding principle64,65,66. Each repeat module amino acid sequence (lower row) of the DBD encompasses the indicated hypervarible diresidues (XX) in positions 12 and 13 that have a capability to bind a specific nucleotide within the target sequence (i.e. the ‘protein-DNA’ code illustrated in the upper two rows). (c) The assembled cDNA sequences for DBDs of TALEN-left and TALEN-right were inserted into indicated sites, respectively. (d) The expression constructs for TALEN-left (6587 bp) and TALEN-right (5425 bp) (upper panel), together with their PstI/BamHI-digested fragments (lower panel), were identified by their electrophoretic mobility on 0.8% agar gel, before being sequenced to ensure the fidelity of the inserted DBDs-coding cDNA fragments. (e) HEK293 cells were co-transfected for 6 h with TALEN-left and TALEN-right constructs (at a ratio of 1:2), along with a GFP expression plasmid. The cells were then selected by treatment with puromycin (2 μg/ml) for 48 h before being subjected to the cloning of single cells grown in 96-well plates, in order to determine the activity of TALENs-mediated gene editing. (f) The genomic DNA from the individual cell clones served as a template of PCR to amplify the TALENs-target region of Nrf1, followed by sequencing of PCR products. The result revealed that overlapped peaks of distinct nucleotide waves started around the specific site responsible for translation of Nrf1α. (g) A nucleotide alignment of the human wild-type (WT) Nrf1 and its frameshift mutants around and within TALENs-target sequences. The deletion nucleotides were indicated by dashed dots. The putative cDNA codons were placed in the black backgrounds.
