Application of CRISPR/Cas9 nuclease in amphioxus genome editing

Abstract The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrate due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) has been adopted in amphioxus genome editing, its labor intensive construction of the TALEN proteins limits its usage in many laboratories. We here report an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data show that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs causes no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at two cell stage, or of Cas9 protein and sgRNAs before fertilization can executes efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations at efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrate that monomerization of sgRNAs via thermal treatment or integration of sgRNAs into a longer backbone could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.