Towards functional genetics in the European amphioxus: efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.

Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transciption factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.