Wannaporn Ittiprasert 1 , 2 , Victoria H Mann 1 , 2 , Shannon E Karinshak 2 , Avril Coghlan 3 , Gabriel Rinaldi 3 , Geetha Sankaranarayanan 3 , Apisit Chaidee 2 , 4 , Toshihiko Tanno 5 , 6 , Chutima Kumkhaek 7 , Pannathee Prangtaworn 2 , 8 , Margaret M Mentink-Kane 9 , Christina J Cochran 1 , Patrick Driguez 3 , Nancy Holroyd 3 , Alan Tracey 3 , Rutchanee Rodpai 4 , Bart Everts 10 , Cornelis H Hokke 10 , Karl F Hoffmann 11 , Matthew Berriman 3 , Paul J Brindley , 1
CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.
Schistosomiasis is a tropical disease that can cause serious health problems, including damage to the liver and kidneys, infertility and bladder cancer. Nearly a quarter billion people are currently infected, mostly in poor regions of sub-Saharan Africa, the Philippines and Brazil.
A freshwater worm known as Schistosoma mansoni causes the disease. These parasites enter the human body by burrowing into the skin; once in the bloodstream, they move to various organs where they rapidly start to reproduce. Their eggs release several molecules, including a protein known as omega-1 ribonuclease, which can damage the surrounding tissues.
A gene editing technique called CRISPR/Cas9 allows scientists to precisely target and then deactivate the genetic information a cell needs to produce a given protein. While the tool has been used in other species before, it was unknown if it could be applied to S. mansoni. Here, Ittiprasert et al. harnessed CRISPR/Cas9 to deactivate the gene that codes for omega-1 ribonuclease and create parasites that do not produce the protein, or only very little of it. The experiments showed that mice infected with the gene-edited worm eggs displayed far fewer symptoms of schistosomiasis compared to those that carry the non-edited parasites.
Alongside this work, Arunsan et al. used CRISPR/Cas9 to inactivate a gene in another species of worm that can cause liver cancer in humans. Together, these findings demonstrate for the first time that the gene editing method can be adapted for use in parasitic flatworms, which are a major public health problem in tropical climates. This tool should help scientists understand how the parasites invade and damage our bodies, and provide new ideas for treatment and disease control.