Schistosome Transgenesis: The Long Road to Success

Abstract

As research on parasitic helminths has entered the post-genomic era, research efforts have turned to deciphering the function of genes in the public databases of genome sequences. It is hoped that, by understanding the role of parasite genes in maintaining their parasitic lifestyle, critical insights can be gained to develop new intervention and control strategies. Methods to manipulate and transform parasitic worms are now developed to a point where it has become possible to gain a comprehensive understanding of the molecular mechanisms underlying host-parasite interplay, and here, we summarise and discuss the advances that have been made in schistosome transgenesis over the past 25 years. The ability to genetically manipulate schistosomes holds promise in finding new ways to control schistosomiasis, which ultimately may lead to the eradication of this debilitating disease.

Keywords: CRISPR; RNA interference; RNAi; functional genomics; genome editing; genomic safe harbour site; mobile genetic elements; schistosomiasis; transfection.

Figure 1

Life cycle of Schistosoma spp. Schistosome eggs are shed into fresh water from infected humans with faeces or urine, depending on species (1): . The first larval stages of the parasite (miracidia) hatch from eggs (2) and seek out and penetrate specific snail intermediate hosts (3). In the snail, the miracidia develop into two consecutive generations of sporocysts (4), which produce large numbers of cercariae, the second larval form of the parasite (5). Cercariae are released from the snail and seek out and penetrate the skin of the human host (6). During penetration. they shed their forked tails to become schistosomulae (7). These juvenile worms migrate via the venous circulation to the lung and heart and finally develop in the liver (8) and (9). Male and female adult worms copulate and finally reside in the mesenteric venules of the bowel/rectum or the venous plexus of the bladder (10) (see , , and for specific locations depending on schistosome species) [10].

Figure 2

Transfection methods used for transgenesis in schistosomes. (A) Particle bombardment (gene gun) for the transfer of expression plasmid-covered gold particles. (B) Two-component piggyBac transposon system. pXL-BacII includes the piggyBac terminal inverted repeats (red arrows), an ampicillin resistance gene, a bacterial ColE1 replication origin, and the S. mansoni actin 1.1 gene promoter, driving the firefly luciferase gene. pBSII-IE1-orf is a piggyBac helper plasmid, providing the piggyBac transposase under the control of the IE1 promotor. After a restriction digest of pXL-BacII and in vitro transcription of the transposase from pBSII-IE1-orf, the IR-Act-Luc-IR cassette and transposase mRNA are transferred into schistosomes via electroporation, leading to luciferase expression. (C) Gene suppression by micro-RNA. The precursor RNA is either delivered into the cell via electroporation or with a lentiviral construct. In lentiviral delivery, DROSHA ribonuclease III cleaves primary miRNA (pri-miRNA) hairpins in the nucleus. The resulting precursor miRNA (pre-miRNA) is exported to the cytoplasm. The pre-miRNA is further processed by DICER to produce mature miRNA duplexes. RISC uses the miRNA as a template for recognising complementary mRNA. When a complementary strand is found, it activates RNase and cleaves the RNA. VSV-G: Vesicular stomatitis virus glycoprotein. (D) Gene editing with CRISPR. After the delivery of the Cas9 protein and sgRNA via electroporation, the target DNA is cut. Non-homologous end-joining leads to a disruption of the targeted gene (knockout), while homology-directed repair leads to gene knock-in with the help of a homology repair donor DNA. Created with BioRender.com.

Figure 3

Transfection targets in schistosome life-cycle stages described in the text. (A) Genes targeted by RNA interference. (B) Genes targeted by gene editing using CRISPR/Cas9. Created with BioRender.com.