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. 2018 May 29;16(12):2053–2062. doi: 10.1111/pbi.12938

Figure 4.

Figure 4

Targeted mutagenesis at the TaGW2 locus using purified TALEN protein. (a) The gene architecture of TaGW2 and the TALEN target site in exon 8. The left and right target sequences are underlined and the spacer sequence is in lower case. The SNP in the target sequence of TaGW2‐D1 is highlighted in green. (b) Diagram of the plasmid used for bacterial expression and purification (left). SDS‐PAGE of the purified GW2‐L and GW2‐R proteins (right). (c) Mutation frequency at the gw2‐TALENs target site in protoplasts detected by the PCR/RNP method using different RNA‐guided endonucleases with a conserved primer set. ‘1’ indicates protoplasts incubated with the gw2‐TALENs protein, and ‘CK’ is a negative control. (d) Mutation frequency induced by purified gw2‐TALENs protein in protoplasts detected by the PCR/T7EI method with homoeologue‐specific primer sets. The red arrows indicate mutant bands. (e) Outcome PCR/RNP to detect TALEN‐induced mutations in 12 representative T0 plants. ‘WT/D’: wild‐type amplicons digested with CRISPR/Cas9 RNP, ‘WT/UD’: wild‐type amplicon not digested.