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.