The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly

• We present an easy protocol for manual selection of TALEN targets.
• We use TALEN RNA with plasmid-encoded poly(A) tails.
• We show two simple TALEN activity evaluation assays based on egg microinjections.
• We show mutation detection procedures based solely on molecular assays.
• Our method often allows the reception of homozygous mutants already in the G1.

Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.