Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

AbstractIdentification, characterization, and transgene insertion into a genomic safe harbor site (GSH) is expected to facilitate consistent transgene activity without disruption to the host cell genome while supporting transgene activity. The location of GSH sites was predicted in the human blood fluke,Schistosoma mansoniusing a computational approach, and after which a CRISPR-focused protocol was developed for insertion of a reporter transgene into a representative GSH. Ribonuclear protein complexes of Cas9 nuclease, three overlapping guide RNAs, and phosphorothioate-modified, double-stranded donor DNA encoding green fluorescent protein driven by a ubiquitously expressed promoter were employed. Gene knockout efficiencies of 24% and reporter knockin transgene fluorescence of >70% of gene-edited schistosome eggs were observed. These outcomes advance functional genomics for schistosomes by providing a tractable path towards transgenic helminths using homology directed repair catalyzed transgene insertion. This approach should be adaptable to helminths generally.