Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

Summary The evolution of animal behaviour is poorly understood 1 , 2 . Despite numerous correlations of behavioural and nervous system divergence, demonstration of the genetic basis of interspecific behavioural differences remains rare 3 – 5 . Here, we develop a novel neurogenetic model, Drosophila sechellia , a close cousin of D. melanogaster 6 , 7 that displays profound behavioural changes linked to its extreme specialisation on noni fruit 8 – 16 . Using calcium imaging, we identify D. sechellia olfactory pathways detecting host volatiles. Mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni. Cross-species allele transfer demonstrates that tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterise their evolutionary origin and behavioural significance. Through circuit tracing in the D. sechellia brain, we find that receptor adaptations are accompanied by increased sensory pooling onto interneurons and novel central projection patterns. This work reveals the accumulation of molecular, physiological and anatomical traits linked to behavioural divergence, and defines a powerful model for investigating nervous system evolution and speciation.