Schistosoma mansoni genome safe harbor editing

The genomic DNA was extracted from the multi-organism S. mansoni parasite after CRISPR/Cas 9 gene editing on gene safe harbor (GSH) with or without DNA donor(s). Briefly, schistosome parasites were delivered with CRISPR component in the ribonucleoprotein complex (RNP) including cas9 enzyme and and guide RNA targeting GSH sites. The transgene donor delivered at the same time with RNP by electroporation. Then, after 10 days, genomic DNAs were extracted and target amplicon were sequenced by Sanger Direct Sequencing prior to confirm the mutation by next generation sequencing. The mutation resulting by CRISPR/Cas were investigated by comparison chromatograms of Sanger direct sequencing data between control and experimental group using TIDE and/or ICE and/or DECODR software analysis. The NGS datas were analyzed by CRISPRressov2 software. In the case of fluorescent protein KI, we also investigate for inserted fluorescent gene transcript driven by parasite gene promoter by RT-PCR from schistosome RNA as well.