The CRISPR-Cas9 system permits researchers to quickly edit genes for functional protein knockout in mammalian, fish and plant genomes, among others, and consequently has dramatically transformed biological research. The CRISPR-Cas9 system requires exogenous Cas9 nuclease to be delivered into the cell, which can be accomplished through transfection of an expression plasmid, mRNA or protein, or through transduction with lentiviral particles. DNA-based Cas9 constructs, while appropriate for many applications, may result in unwanted integration events. Lentiviral delivery results in integration of the Cas9 expression cassette into the cell’s genome, and transfection of a Cas9 plasmid may result in the insertion of vector sequence at the site of the double-stranded break when the genomic DNA is repaired through the NHEJ pathway. The use of Cas9 mRNA or protein avoids any unwanted integration, and in combination with using synthetic crRNA and tracrRNA, results in a completely DNA-free gene editing system. Here we demonstrate successful gene editing using DNA-free CRISPR-Cas9 reagents for gene knockout in zebrafish. Zebrafish embryos from a stable transgenic line were injected with Cas9 nuclease mRNA, synthetic tracrRNA, and crRNA designed to target GFP. A mismatch detection assay confirmed efficient gene editing, and successful functional protein knockout was confirmed by loss of GFP fluorescence.