Figure 4.

Recovery of targeted modifications without selection or enrichment. A, Flow diagram of the experimental protocol. B, DNA sequences of NHEJ-induced mutations in ALS recovered from three representative calli derived from protoplasts transformed with TALEN T30. All three calli have one allele each of SurA and SurB modified, as revealed by distinctive DNA sequence signatures (underlined residues). Sequences in lowercase denote spacers between the two TALEN binding sites. C, Schematic of the gene-targeting experiment using a donor molecule that modifies 6 bp of coding sequence; the modifications create an XhoI restriction site. Arrowheads denote PCR primers used in the molecular characterization of recombinants. ORF, Open reading frame. D, Molecular analysis of independent calli derived from treatment with TALEN T30 and the donor molecule depicted in C. A region of the coding sequence was PCR amplified (arrowheads in C) and digested with XhoI. Lanes 3 to 20 are the digestion products from 18 randomly chosen calli. Lanes 1 and 2 are products from calli treated with the donor only or TALEN T30 only, respectively. M, Molecular length markers. E, DNA sequences of recombinants from lanes 5 and 14 in D. Underlined sequences in lowercase denote base changes introduced into the coding sequence by homologous recombination.