Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Abstract

34 35 CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested 36 this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This 37 secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative 38 immuno-pathological readouts for programmed genome editing. Schistosome eggs were either 39 exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary 40 to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and 41 the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor 42 transgene that encoded six stop codons, flanked by 50 nt-long 5'-and 3'-microhomology arms 43 matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso 44 analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, 45deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion 46 of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ω1-edited 47 eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the 48 ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including 49 IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines 50followed the exposure to those of ω1-mutated schistosome eggs. Following injection of 51 schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas 52surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome 53 editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by 54 homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the 55 capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of 56 pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype 57showcase the novel application of programmed gene editing in and functional genomics for 58 schistosomes. 59 60 61 Introduction 62 63Schistosomiasis is considered the most problematic of the human helminth diseases in terms of 64 morbidity and mortality [1][2][3][4]. The past decade has seen major advances in knowledge and 65 understanding of the pathophysiology, developmental biology, evolutionary relationships and 66 genome annotation of the human schistosomes [5][6][7][8][9][10][11][12][13][14][15][16]. Establishing CRISPR/Cas9 genome 67 editing in schistosomiasis would greatly enable effective functional genomics approaches. The 68 stable CRISPR/Cas9-based site-specific gene mutation and phenotyping will drive innovation 69 and a deeper understanding of schistosome pathogenesis, biology and evolution [17]. 70 71The schistosome egg plays a central role both in disease transmission and pathogenesis [1]. The 72 appearance of S. mansoni eggs in host tissues by six to seven week...

Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signalling and drug detoxification and are essential for parasite survival

“…The smche transcript levels of silenced worms recovered from mice were no different from control parasites and it is likely that the surviving worms received less siRNA than those worms that died in the host. Alternatively, surviving worms might have recovered from the transient effects of RNAi, highlighting the advantage of targeted gene knockout techniques such as CRISPR/Cas9 which has recently been reported for the first time in schistosomes [59].…”

The Transcriptome of Schistosoma mansoni Developing Eggs Reveals Key Mediators in Pathogenesis and Life Cycle Propagation

“…Thus, numerous reports have focused on soluble egg antigens (SEA) and several excreted-secreted products, including proteins and glycans that interact with the host tissues, inducing immune responses and facilitating the egress of the egg to the external environment (10)(11)(12)(13)(14). Notably, the immune modulatory roles of egg-specific antigens, such as Omega, Kappa and IPSE have been validated by functional approaches such as shRNA-mediated knock-down (15) and CRISPR-Cas-based genome editing (16). More recently, the molecular and cellular mechanisms involved in granuloma formation have been dissected using a zebrafish model of macrophage dependent granuloma induction (17).…”

Novel cholinesterase paralogs ofSchistosoma mansonihave perceived roles in cholinergic signaling, glucose scavenging and drug detoxification and are essential for parasite survival

“…The smche transcript levels of silenced worms recovered from mice were no different from control parasites and it is likely that the surviving worms received less siRNA than those worms that died in the host. Alternatively, surviving worms might have recovered from the transient effects of RNAi, highlighting the advantage of targeted gene knockout techniques such as CRISPR/Cas9 which has recently been reported for the first time in schistosomes [61].…”