**5. Zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALEs)**

Although many methods have been developed, soybean is considered a recalcitrant plant to transform compared to *Arabidopsis* and rice. Since full genome sequencing data has been rapidly updated in soybean, soybean transformation technology is becoming an essential approach for genomic research. For the phenotypic analysis of genes, knock-out or gene-si‐ lencing plants are used to study gene function. However in soybean, making bulk knock-out mutants through conventional mutagenesis approaches is not immediately feasible because of low transformation efficiency. Thus, development of innovative gene targeting methods is necessary to make knock-out plants in soybean.

Zinc finger nucleases (ZFNs) and meganucleases cut specific DNA target sequences *in vivo* and thus are powerful tools for genome modification. In particular zinc finger domain, which predominantly recognize nucleotide triplets, have been widely used in this research. Importantly, ZFNs modification has been reported in soybean (*Glycine max*), maize (*Zea mays*), tobacco (*Nicotiana tabacum*) and *Arabidopsis* [87-90]. Unfortunately, Ramirez et al. [91] found a major disadvantage of ZFNs; they observed that the modular assembly method of engineering zinc-finger arrays has a higher failure rate than previously reported.

To overcome the ZFNs's weakness, in late 2009, a novel DNA binding domain was identi‐ fied which was a member of the large transcription activator-like (TAL) effector family [92-93]. Transcription activator-like effectors (TALEs) are produced by plant pathogens in the genus *Xanthomonas* as virulence factors and TAL effector–mediated gene induction leads to plant developmental changes [94-95]. The type III secretion system is used by *Xanthomo‐ nas* to introduce virulence factors into plant cells [96]. Once inside the plant cell, transcrip‐ tion activator-like (TAL) effectors (TALEs) enter the nucleus, bind effector-specific DNA sequences, and transcriptionally activate gene expression [97-98]. For genomic engineering, two methods of TALEs were developed: TALE nucleases (or TALENs) and TALE transcrip‐ tion factors (or TALE-TFs). Both TALENs and TALE-TFs contain as many as 30 tandem re‐ peats of a 33- to 35-amino-acid-sequence motif (Figure 2). The amino acids in positions 12 and 13 in each 33- to 35-amino-acid-sequence motif have the repeat variable di-residue (RVD). Using this specific ability, two pair (left and right TALENs) of repeats with different RVDs are designed by PCR and bound in the target DNA sequence [92-93]. Fok1 combined with TALE nucleases (or TALENs) make double-strand breaks (DSBs) at specific locations in the genome. These DSBs are repaired by homologous recombination (HR) or non-homolo‐ gous end-joining (NHEJ) pathways. During DSBs repair, errors in genome via insertion, de‐ letion, or chromosomal rearrangement could be induced by HR and NHEJ (Figure 2). Unlike TALENs, TALE-TFs require only a single TALE construct for activity induction when com‐

bined with VP64 activator (derived from the herpex simplex virus activation domain). VP64 binds with RNA polymerase and causes transcriptional activation of the gene of interest [99]. For making transgenic plants, the TALEs technique can be combined with the *Agrobac‐ terium*-mediated transformation method and it is assumed that gene targeting knock-out will be applied in soybean research.

**Figure 2.** Summary of Transcription activator-like effectors (TALEs) nuclease.
