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 a 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.
Database accessions Sequence reads from the amplicon NGC libraries are available at the European Nucleotide Archive, study accession number ERP110149. Additional information is available at Bioproject PRJNA415471, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA415471 and GenBank accessions SRR6374209, SRR6374210.
Sequence reads from the amplicon NGC librariesEuropean Nucleotide Archive, ERP110149.
Schistosoma mansoni gene editing by CRISPR/Cas 9 targeting omega-1 , exon 1NCBI BioProject, PRJNA415471.
- Karl F Hoffmann
- Gabriel Rinaldi
- Apisit Chaidee
- Pannathee Prangtaworn
- Matthew Berriman
- Paul J Brindley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Mice experimentally infected with S. mansoni, obtained from the Biomedical Research Institute (BRI), Rockville, MD were housed at the Animal Research Facility of the George Washington University Medical School, which is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC no. 000347) and has an Animal Welfare Assurance on file with the National Institutes of Health, Office of Laboratory Animal Welfare, OLAW assurance number A3205-01. All procedures employed were consistent with the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) of the George Washington University approved the protocol used for maintenance of mice and recovery of schistosomes. Studies with BALB/c mice involving tail vein injection of schistosome eggs and subsequent euthanasia using overdose of sodium pentobarbital was approved by the IACUC of BRI, protocol 18-04, AAALAC no. 000779 and OLAW no. A3080-01.
- James B Lok, University of Pennsylvania, United States
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.