Amphioxus, also called lancelet or cephalochordate, is a promising model organism owning to its particularly evolu- tionary position, simple genome content and comparable body plan to that of vertebrates (Holland et aL, 2004; Bertrand and Escriva, 2011). However, use of amphioxus as a model or- ganism has been limited for many years because of lack of an efficient genomic modification method. Recently, several revolutionary gene targeting methods that could induce directed mutations, insertions and deletions at intended target sites, have been developed (Gaj et al., 2013).