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Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the  B. mori  genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the  B. mori  embryo. We also established a novel  B. mori  somatic cell assay suitable for the preselection of highly efficient TALENs directly in the  B. mori  model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the  BmBLOS2  gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene,  red egg  ( Bm-re ) and showed that it allows obtaining homozygous mutants in G 1 . Our procedure minimizes the chance of failure in  B. mori  gene targeting experiments.

Efficient TALEN Construction for Bombyx mori Gene Targeting