Figure - available from: Frontiers in Immunology
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CRISPRi of SmfgfrA in S. mansoni eggs. (A) SmfgfrA mRNA level in eggs treated with i4 or i5. WT eggs and NC treated eggs were used as controls. (B) Transcription level of SmfgfrA in miracidia hatched from WT eggs and eggs treated with NC, i4 or i5. (C) Effects of SmfgfrA repression on the hatching of S. mansoni eggs. The hatching efficiency (%) of WT eggs and eggs treated with NC, i4 or i5 was calculated by dividing the number of hatched eggs with the total number of eggs (hatched and unhatched) X 100. Experiments in panels (A–C) was performed in triplicates with all data presented as the mean ± SE. (D) Western blot showing expression level of SmFGFRA protein in SEA of WT eggs (Lane 1) and eggs treated with NC (Lane 2), i4 (Lane 3) or i5 (Lane 4). An anti-actin antibody was utilized as control to ensure equal loading. The electrophoresed SEAs were transferred for the western blot analysis to two PVDF membranes which were probed simultaneously with the anti-rSmFGFRA-L antibody (left blot) and the anti-actin antibody (right blot). (E) Caspase-3/-7 activity in soluble egg antigen (SEA) of WT eggs and eggs treated with NC, i4 or i5. This assay was conducted in duplicate with all data presented as the mean ± SE. * p value≤ 0.05, ** p value≤ 0.01, *** p value ≤ 0.001.
Source publication
Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells a...
Citations
... Indeed, exposure of adult stage schistosomes to CRISPR by electroporation can be expected to result in genetic mosaicism. Our findings conformed with earlier reports of deletion mutations at CRISPR targets in LE, schistosomula, and adult schistosomes (26,61,87,94,95). Nonetheless, the outcome can be informative for proof-of-principle studies of genome editing or as an alternative for RNAi of genes transcribed in the (sub)tegument. ...
Recently, we reported programmed Cas9 mediated insertion of a reporter gene into a gene safe harbor site, GSH1, of Schistosoma mansoni via homology-directed repair (HDR) using overlapping guide RNAs. Here, we report efficient and precise CRISPR/Cas12a-mediated homology-directed insertion of a 5’ C6-PEG10-modified double-stranded transgene bearing microhomology arms, 50 nt length at GSH1. At the outset, we undertook bioinformatic and computational analysis following by experimental verification of the regulatory activity of schistosome ubiquitin (SmUbi) promoter and terminator, aiming to drive strong reporter gene expression. Green fluorescent protein activity driven by this endogenous promoter followed electroporation-mediated transfection of schistosome eggs. HDR induced by CRISPR/Cas12a delivered more efficient transgene integration compared to CRISPR/Cas9, with precise integration of the transgene at Cas12a cleavage sites, which releases overhanding DNA strands at 18-24 nt during programmed cleavage. The transfection design for CRISPR/Cas-mediated genome editing facilitated precise knock-in, specifically chromosomal integration of the SmUbi-EGFP-SmUbi reporter-gene with microhomology arms into GSH1. In this non-model system, the 5’-C6-PEG10 modified-DNA template enhanced knockin efficiency of Cas9 and Cas12a. This approach advances schistosome transgenesis and may be functional also in related parasitic and non-parasitic helminths, which hitherto lack functional genomics and transfection methods.
GRAPHICAL ABSTRACT
... 18,37 Overlapping gRNAs rather than simply multiple gRNA may be more efficient for gene KO in S. mansoni given recent findings involving CRISPR interference that compared both single and multiple gRNAs. 18,19,38 GSH1 represents a promising CRISPR target for S. mansoni. Notably, 75% of eggs exhibited EGFP in the miracidium developing within the eggshell and significantly more fluorescence than seen in the control eggs transfected only with donor template. ...
The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.
Control of schistosomiasis japonica, endemic in Asia, including the Philippines, China, and Indonesia, is extremely challenging. Schistosoma japonicum is a highly pathogenic helminth parasite, with disease arising predominantly from an immune reaction to entrapped parasite eggs in tissues. Females of this species can generate 1000–2200 eggs per day, which is about 3- to 15-fold greater than the egg output of other schistosome species. Bovines (water buffalo and cattle) are the predominant definitive hosts and are estimated to generate up to 90% of parasite eggs released into the environment in rural endemic areas where these hosts and humans are present. Here, we highlight the necessity of developing veterinary transmission-blocking vaccines for bovines to better control the disease and review potential vaccine candidates. We also point out that the approach to producing efficacious transmission-blocking animal-based vaccines before moving on to human vaccines is crucial. This will result in effective and feasible public health outcomes in agreement with the One Health concept to achieve optimum health for people, animals, and the environment. Indeed, incorporating a veterinary-based transmission vaccine, coupled with interventions such as human mass drug administration, improved sanitation and hygiene, health education, and snail control, would be invaluable to eliminating zoonotic schistosomiasis.
