Abstract
We identified genomic safe harbor sites (GSH) in the human blood fluke, Schistosoma mansoni and developed a CRISPR-focused protocol for insertion of a reporter transgene into a representative GSH. The protocol employed ribonuclear protein complexes of Cas9 nuclease, three overlapping guide RNAs, and phosphorothioate-modified, double stranded donor DNAs encoding green fluorescent protein driven by a strong endogenous promoter. Gene-editing efficiencies of >20% and reporter transgene fluorescence of >50% of gene-edited eggs were obtained by five days after CRISPR transfection. These methods and results advance functional genomics for multicellular parasites, and represent a tractable path towards transgenic schistosomes using homology directed repair catalyzed transgene insertion. Identification and characterization of GSH is expected to facilitate consistent transgene activity with neutral influence on the host cell genome and, concurrently, provide a privileged locus for transgene activity. This approach should be adaptable to platyhelminths generally.