@article {Ittiprasert358424, author = {Wannaporn Ittiprasert and Victoria H. Mann and Shannon E. Karinshak and Avril Coghlan and Gabriel Rinaldi and Geetha Sankaranarayanan and Apisit Chaidee and Toshihiko Tanno and Chutima Kumkhaek and Pannathee Prangtaworn and Margaret Mentink-Kane and Christina J. Cochran and Patrick Driguez and Nancy Holroyd and Alan Tracey and Rutchanee Rodpai and Bart Everts and Cornelis H. Hokke and Karl F. Hoffmann and Matthew Berriman and Paul J. Brindley}, title = {Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke, Schistosoma mansoni}, elocation-id = {358424}, year = {2018}, doi = {10.1101/358424}, publisher = {Cold Spring Harbor Laboratory}, abstract = {CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5{\textquoteright}-and 3{\textquoteright}-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed \~{}4.5\% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19\% insertion of the donor transgene. Transcripts encoding ω1 were reduced \>80\% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.}, URL = {https://www.biorxiv.org/content/early/2018/08/17/358424}, eprint = {https://www.biorxiv.org/content/early/2018/08/17/358424.full.pdf}, journal = {bioRxiv} }