Genetic manipulations in helminth parasites

Abstract

Screening of vaccine or drug target in parasitic helminth is hindered by lack of robust tool for functional studies of parasite protein which account for the availability of only a few anti-helminthic vaccines, diagnostic assay and slower pace of development of an anthelmintic drug. With the piling up of parasite transcriptomic and genomic data, in silico screening for possible vaccine/drug target could be validated by functional characterization of proteins by RNA interference or CRISPR/Cas9. These reverse genetic engineering tools have opened up a better avenue and opportunity for screening parasitic proteins in vitro as well as in vivo. RNA interference provides a technique for silencing targeted mRNA transcript for understanding a gene function in helminth as evidence by work in Caenorhabditis elegans. Recent genetic engineering tool, CRISPR/Cas9 allows knock-out/deletion of the desired gene in parasitic helminths and the other provision it provides in terms of gene knock-in/insertion in parasite genome is still to be explored in future. This manuscript discussed the work that has been carried out on RNAi and CRISPR/Cas9 for functional studies of helminth parasitic proteins.

Introduction

Parasitic helminthiasis pose a relentless constrain to livestock performances in terms of reduction in growth, wool and milk production and even mortality of animal as well (Mehra et al. 1999; Charlier et al. 2007; Brockwell et al. 2014). Due to cheap, easily available and wide safety index, there is indiscriminate use of antiparasitic compound in livestock causing the emergence of parasites with low or no efficacy against the administered drug (Sanyal et al. 2008; Chandra et al. 2015). The control of parasitic infections in a population, the supra-population control: through intermediate host control, grazing management in pasture, the use of biological control and other novel control regime is questionable to challenge these menaces as a replacement for chemotherapy (Jackson and Miller 2006; Gordon et al. 2012). It is a need of the hour to come up with effective immuno-prophylactic measures as chemotherapy pose additional concern such as drug residues in meat, milk and egg along with those excreted in faeces contaminating the environment (Gordon et al. 2012; Hanna et al. 2015).

Several factors have contributed to the slow progress in parasitic vaccine development, such as: the colossal size of parasite genome (for example, Haemonchus contortus, which is 370 Mbp) makes an arduous task for mining a gene/protein responsible for parasite vital functions or pathogenesis (Laing et al. 2013); lack of understanding of host immunity against parasites, i.e., the host-parasite interplay (Molina-Hernandez et al. 2015); complexity of parasitic protein/enzymes which vary their expression depending on the stage of parasite, for example, Fasciola secretes at least 13 types of cysteine proteinases among which cathepsin Bs are express predominantly in immature where cathepsin Ls in adult parasite (Dalton et al. 2003; Celias et al. 2019); elegant mode of evasion of immunity by parasite such as glycocalyx turn over by Fasciola (Hanna et al. 1980); dampening the host immunity via production of adenosine by Schistosoma mansoni (Bhardwaj and Skelly 2011); functional studies of parasitic protein for vaccination studies face stumbling block such as difficulty in prolonged culture of parasite in vitro (Lalrinkima 2016) and lack of proper definition of the target antigen could in part resulted in partial understanding of the host immune response during vaccination studies (Maggioli et al. 2011; Raina et al. 2011).

Parasite genomic and proteomic studies have opened up new frontiers of knowledge on parasite biology, host-parasite interactions, improved diagnostics and possible control measures against these pathogens (McVeigh and Maule 2019). Traditionally, vaccination studies are carried by purifying immuno-dominant protein after screening the reactivity of these antigens with infected sera of an animal. For instance, screening of somatic protein or excretory/secretory protein of Fasciola gigantica is carried out after an antigen is subjected to immuno-reactivity with infected buffalo serum in western blot (Dixit et al. 2002; Gupta et al. 2003). In the case of ectoparasite such as tick, the parasitic proteins are subjected to series of affinity chromatography with each fraction is subjected for vaccination trial yielding Bm86 after many trials and errors (Willadsen et al. 1988). This kind of approach is not only prone to error with inconsistent results but expensive and time-consuming requiring several years to standardise the protocols as well as batch-to-batch variation of the isolated proteins (Willadsen et al. 2006). The advancement in molecular biology in recent times has piled up parasite genomic data that allows screening for potential vaccine candidate in silico (Howe et al. 2017). Functional studies of parasite protein for vaccine antigen or drug target by gene silencing through RNA interference and gene knock out by CRISPR-Cas9 in recent years allows an understanding of protein discrete function with high fidelity and subsequent validation (McVeigh et al. 2019; Dalzel et al. 2012). These approaches will likely minimise the time of vaccination studies, experimental errors and financial expenditure as well especially in neglected tropical parasitic diseases.

Parasite genomics

It has now become possible to sequence the whole genome of key parasites which represents a tremendous resource for research on parasites. Genome sequences will serve as the basis for future functional analyses of the newly discovered genes but the progress of research studies on parasites with no human or zoonotic importance is rather slow (Blake 2015). The use of new long-read sequencing techniques rather than assembling the repetitive regions using short sequencing reads could result in improved parasite genome sequences (Talavera-Lopez and Andersson 2017). This information will provide the basis for designing novel vaccines, diagnostic antigen and antiparasitic drugs. Several genomics projects on veterinary parasites like Cryptosporidium parvum (Abrahamsen et al. 2004), Theileria (Gardner et al. 2005), Babesia (Brayton et al. 2007), Toxoplasma gondii (Bontell 2009), Haemonchus contortus (Laing et al. 2013), Eimeria (Reid et al. 2014) and Fasciola sp. (Cwiklinski et al. 2015; Pandey et al. 2020) are completed. In turn, proteins that are responsible for virulence, pathogenesis, host specificity, induction of protective immune responses and proteins that are essential components of metabolic processes of the parasite will be identified (Howe et al. 2017). The expanding genomic and expressed sequence tag (EST) datasets for parasitic flatworms including Schistosoma (Fung et al. 2002; Oliveira 2007; Phuphisut et al. 2018) and Fasciola (Ryan et al. 2008; Zhang et al. 2019) have helped highlight the need for technologies to aid their exploitation. The introduction of next-generation sequencing (NSG) benefit parasitologists with an expansion of approaches for further analysis of parasite genome in terms of comparative genomics, transcriptomics, proteomics, epigenetics etc. which will likely address the problems of parasite epidemiology, drug resistance, fragmentary knowledge of host-parasite interaction, specific gene function etc. (Fire et al. 1998; Vanhove et al. 2018).

Genetic manipulations in parasites

Genomic tools that help to dissect the role of the 134 parasite genes are now required to effectively exploit these datasets. Until recently, genetic manipulations were not standardised properly on parasitic worms and this has repressed progress associated with the elucidation of gene function in helminth parasites. The rapid progress of genomic and gene manipulation techniques will likely result in the emergence of techniques for interrogation and modification of genomes, leading to a better understanding of parasite biology within the host and ultimately to the rational design of improved methods for control of animal parasitic diseases (McVeigh and Maule 2019). Genetic manipulations of parasites are mainly carried out through RNAi and CRISPR-Cas9 which are discussed in the following.

RNA interferences

RNA interference (RNAi) is a biological process of degradation of target mRNA by the introduction of the corresponding RNA of the target sequence, which was historically known by other names, including co-suppression, post-transcriptional gene silencing (PTGS) and quelling. It has been evident that RNAi technology has immense potential in suppression/silencing of desired mRNA degradation based on work on RNA interference in the nematode worm Caenorhabditis elegans (Fire et al. 1998). Earlier, Napoli et al. (1990) attempted to produce a petunia flower with a deeper colour by inserting the gene for purple pigment into its genome. Instead of turning the flower into dark purple, the petunias were either entirely white or streaked purple and white. The authors surmised that the additional copy of the gene suppressed the gene expression itself, and its endogenous counterpart and called co-suppression. Later, Fire et al. (1998) explained that the inserted piece of gene produced a RNA product that interfered with the corresponding target gene thereby its expression in C. elegans worm. The RNAi pathway is found in many eukaryotes, including parasites and the process is initiated as the introduction of double stranded RNA (dsRNA) that is cleaved and process by an enzyme known as Dicer into short double-stranded fragments of ~ 21 nucleotide small interfering RNAs (siRNA). Each double stranded siRNA is unwound into single-stranded RNAs (ssRNAs) as the passenger strand (sense strand) and the guide strand (antisense strand). The passenger strand is degraded subsequently and the guide strand is incorporated into the RNA-induced silencing complex (RISC). When the guide strand pairs with a complementary sequence in the messenger RNA molecule it induces cleavage of itself by Argonaute 2 (Ago2), the catalytic component of RISC thereby degrade the target mRNA for gene silencing (Fig. 1). The discovery of RNAi based gene silencing in free living nematode, Caenorhabditis elegans (Fire et al. 1998) has spur an opportunity to carry out reverse genetic approaches of studying parasite gene function, especially for drug target and vaccine molecule.

Fig. 1.

Schematic diagram of RNA interference using dsRNA [Pictorial representation is designed by using online software (Biorender)]

In parasitic helminth, a certain molecules causing modulation of the immune responses have been identified, leading to the suppression of the host Th1 immune response or mixed type Th responses (Skelly et al. 2003; Valero et al. 2017). The role of these molecules in the parasite metabolism, immune evasion of the host and the host tissue penetration has not been well established in flukes like schistosome as well as in Fasciola. RNAi represents a viable method for understanding the role of these molecules in the parasite survival and its establishment in the host. The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. There is a need to rapidly extend functional genomics approaches to other parasites responsible for several chronic parasitic diseases.

Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNAi reported in several nematodes and trematodes (Fire et al. 1998; Skelly et al. 2003; McGonigle et al. 2008; Stefanic et al. 2010). Since classical reverse genetic approaches remain unavailable in trematodes, RNA interference is the current tool of choice for functional analysis of genes. This approach has been optimized in schistosomes and few other trematodes like Fasciola, Opisthorchis viverrini and Clonorchis sinensis (Rinaldi et al. 2008; Wang et al. 2014; Papatpremsiri et al. 2015; Anandanarayanan et al. 2017). RNAi is a method for selectively silencing or reducing the expression of mRNA transcripts, an approach that can be used to interrogate the function of genes and proteins that enables the validation of potential targets for anthelmintic drugs or vaccines, by investigating the impact of silencing a particular gene on parasite survival or behaviour.

RNAi in flukes

Functional and direct assessment of gene function in parasitic helminth is largely hindered by the non-availability of established functional and reverse genetic engineering tool. Even though RNAi pathway is present in a diverse range of eukaryotic organisms and has permeated all fields of eukaryotic biology as a central technology of molecular analysis by targeted gene silencing, it is largely unexploited in parasites that are not amenable to classical genetic approaches. In parasitic helminth, RNAi studies for gene silencing were carried out in blood fluke, Schistosoma mansoni with great success. Silencing of corresponding cathepsin B in S. mansoni using double-stranded RNA molecule yields suppression of corresponding mRNA transcript as evident by an inability to detect the corresponding gene as well as cathepsin B protein in post-RNAi assessment (Stefanic et al. 2010). Further, target reduction of glucose transporter protein in S. mansoni was achieved as 70–80% reduction of mRNA transcript and 40% reduction of glucose uptake by the parasite (Boyle et al. 2003). Silencing the genes coding for leucine aminopeptidases (LAPs) in the eggs of S. mansoni by RNAi has confirmed the role of LAP in the egg hatching process in the schistosomes. Genetic silencing by RNAi of parasite multidrug resistance transporters disrupts egg production in Schistosoma mansoni (Rinaldi et al. 2009).

In conventional RNAi approaches, dsRNAs or siRNAs are usually delivered to the parasite tissue by soaking, electroporation or through liposome carrier. The post-introduction processing of dsRNAs and longer hairpin RNAs (125–500 bp) within the parasite tissue results in multiple siRNA sequences (21–25 nt) that can target different regions of the mRNA. Synthetic siRNAs can induce target gene knockdown at efficiency comparable with dsRNAs. However, the effectiveness of predicted siRNA requires experimental validation (Ndegwa et al. 2007). The need to validate siRNAs before experimentation might be circumvented and the knockdown effect increased by using siRNA cocktails containing multiple target-specific siRNAs (Zhang et al. 2004; Grimm and Kay 2007). This approach has been utilized to achieve the target gene knockdown in S. mansoni (Beckmann and Grevelding 2012). Although the use of different transfection reagents does not usually improve RNAi efficiency, electroporation can enhance knockdown effects compared with soaking, presumably because more silencing RNA (dsRNA) enters the cells (Correnti et al. 2005; Krautz-Peterson et al. 2007; Ndegwa et al. 2007). Most studies employing RNAi have focused on investigating genes expressed by adult schistosomes, schistosomula and miracidia (Table 1). Target silencing in schistosome through RNAi included genes involved in development e.g. CD36-like scavenger receptor B and glucose-transporter (Dinguirard and Yoshino 2006); haemoglobin digestion (cathepsins B, C, D and asparaginyl endopeptidase) (Skelly et al. 2003; Correnti et al. 2005; Delcroix et al. 2006; Krautz-Peterson et al. 2007; Morales et al. 2008; Stefanic et al. 2010; Tchoubrieva et al. 2010); membrane proteins (Morales et al. 2008; Beckmann and Grevelding 2012) and components of the transforming growth factor TGF-b and spleen tyrosine kinase signalling or redox pathways (Osman et al. 2006; Kuntz et al. 2007; Krautz-Peterson and Skelly 2008; Beckmann and Grevelding 2012).

Table 1.

RNA interference done in various trematode parasites

SI. No	Parasite	Target gene	Types of silencing RNA	Mode of introduction of RNA	Target effect	Authors
1	Schistosoma mansoni	Cysteine protease cathepsin B	dsRNA	Soaking and Liposome	Silencing effect at transcription and translational level	Skelly et al. (2003)
2	-do-	Glucose Transporter Protein 1	dsRNA	Soaking	Target reduction of 70–80% Functional phenotypic reduction of GTP protein (40%)	Boyle et al. (2003)
		GAPDH
3	-do-	Cysteine protease cathepsin B1	dsRNA	Electroporation	Growth retardation	Correnti et al. (2005)
4	-do-	Scavenger Receptor class B (SRB)	dsRNA	Soaking	Target reduction of SRB by 60–70% Reduction of sporocysts binding acetylated LDL Growth retardation of sporocyst	Dinguirard and Yoshino (2006)
		Glucose Transporter Protein 1
5	-do-	Transforming growth factor-beta (TGF-β)	siRNA	Electroporation	Silencing effect at transcriptional level Reduction of GCP expression	Osman et al. (2006)
6	-do-	Cysteine protease cathepsin B	dsRNA and siRNA	Soaking, liposome and electroporation	Electroporation is more effective than soaking	Krautz-Peterson et al. (2007)
7	-do-	Leucine Aminopeptidase (LAP 1 & 2)	dsRNA	Soaking	Silencing effect at transcription and translational level by inhibition of egg hatching	Rinadi et al. (2009)
8	Fasciola hepatica	Cysteine protease cathepsin B and L	dsRNA	Soaking	Silencing at transcription and translational level Reduction of NEJ penetration of intestinal wall	McGonigle et al. (2008)
9	-do-	Leucine aminopeptidase	dsRNA	Soaking	Silencing at transcriptional level Establish the presence of an intact RNAi pathway	Rinaldi et al. (2008)
10	-do-	Cathepsin L, B and Glutathione transferase	dsRNA	Soaking	Silencing at transcription and translational level	McVeigh et al. (2014)
11	Fasciola hepatica	Leucine aminopeptidase	dsRNA and siRNA	Soaking and Electroporation	Target reduction of by 90% dsRNA induces persistence silencing	Dell’Oca et al. (2014)
12	F. gigantica	Superoxide dismutase, Cathepsin L-1 D, B1/3 and B2	dsRNA	-do-	Silencing effect at transcriptional level	Lalrinkima (2016)
13	-do-	Superoxide dismutase, GST, Cathepsin L-1 D, B1/3 and B2	dsRNA	-do-	Silencing effect at transcriptional level	Anandana-rayanan et al. (2017)

The eggs laid by S. mansoni failed to fully mature after soaking with dsRNA targeting the inhibin/activin gene, encoding a member of the TGF-β protein superfamily in vitro (Freitas et al. 2007). Rinaldi et al. (2009) demonstrated an 80% reduction in egg hatching upon knockdown of a leucine aminopeptidase gene. Thus, studies conducted have shown clearly that RNAi can be used effectively for functional genomics investigations and to identify essential genes in Schistosoma. The silencing effect after an introduction of synthetic siRNAs and dsRNAs into S. mansoni persists for several weeks in vitro (Correnti et al. 2005; Krautz-Peterson et al. 2007; Hagen et al. 2014).

Mourao et al. (2009) demonstrated a specific and consistent RNAi-mediated silencing of antioxidants like Glutathione-S-transferase (GST) 26 and 28, Peroxiredoxin (Prx) 1 and 2 and Glutathione peroxidase (Gpx) transcripts but an unexpected elevation of Superoxide dismutase (SOD) transcripts in sporocysts treated with gene-specific dsRNA. The killing of S. mansoni sporocysts by haemocytes of snails was increased in larvae treated with dsRNA of Prx1/2, GST26 and GST28, compared to those treated with SOD dsRNAs in vitro. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions.

Traditionally, functional studies of liver fluke proteins were hindered by a relatively lack of nucleotide sequence datasets compared to other parasitic helminths. However, the ongoing efforts have seen primarily the adult-derived sequences complemented by transcriptome datasets for adult species of both F. hepatica and F. gigantica complemented by juvenile transcriptome datasets and draft F. hepatica/F. gigantica genome sequences (Cwiklinski et al. 2021; Ilgová et al. 2022). The existence of viable and functional RNAi pathway in Fasciola hepatica has opened the gates for functional genomics studies in Fasciola (McGonigle et al. 2008; Rinaldi et al. 2008). In F. hepatica, RNAi has helped to understand the role of cathepsin-L cysteine proteinase in the penetration of gut epithelium by newly excysted juveniles of F. hepatica through a gut wall of rat during its invasion process of the liver (McGonigle et al. 2008). In case of tropical liver fluke, F. gigantica, RNAi methodology was optimized showing efficient and persistent gene knockdown that opens the prospect for functional studies in tropical liver fluke (Lalrinkima 2016; Anandanarayanan et al. 2017). Targeting a single-copy gene encoding leucine aminopeptidase (LAP) as a model, the delivery of silencing RNA was redefined as electro-soaking, i.e. electroporation and subsequent incubation was found to be efficient for the introduction of small RNAs into the fluke (Dell’Oca et al. 2014). Robust transcriptional silencing of multiple genes was standardised in F. hepatica and F. gigantica NEJs where transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence supporting the development of multiple-throughput phenotypic screens for control target validation (McVeigh et al. 2014; Anandanarayanan et al. 2017). While both long dsRNA and short interfering RNA (siRNA) were equally effective at inducing a short-term knockdown, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in Fasciola (Dell’Oca et al. 2014).

Functional genomics was not reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis until Sripa et al. (2011) described the introduction of Cy3-labeled siRNA into adult O. viverrini worms. Adult flukes were subjected to the introduction of siRNA and dsRNA by electroporation resulted in a significant reduction in specific mRNA levels encoding cathepsin B and a significant reduction in cathepsin B activity. RNAi experiment in C. sinensis was also carried out to confirm the biological importance of enolase enzyme by the introduction of dsRNA by soaking approach which resulted in penetration of dsRNA into adult worms and metacercariae. The introduction of dsRNA effectively down-regulate the expression of Csenolase in both adult parasites and metacercariae resulting into killing of C. sinensis adult worms (Wang et al. 2014).

RNAi in nematode

RNA interference in animal parasitic nematode has been reported in adult and larval parasites with varying successes (Table 2). Unlike trematode parasites, RNAi in nematode is seen with a lack of visible phenotypic or morphological changes and variation in susceptibility of certain target genes, besides, some worm like Heligmosomoides polygyrus, murine intestinal nematode, are refractile to RNAi pathways (Lendner et al. 2008). RNAi has been validated in Nippostrongylus braziliensis (Hussein et al. 2002), Brugia malayi (Aboobaker and Blaxter 2003; Ford et al. 2009; Song et al. 2010; Misra et al. 2017), Onchocercus volvulus (Lustigman et al. 2004; Ford et al. 2005), Trichostrongylus colubriformis (Issa et al. 2005), Oestertagia ostertagi (Visser et al. 2006), Haemonchus contortus (Geldhof et al. 2006; Kotze and Bagnall 2006; Samarasinghe 2010; Naqvi et al. 2020) and Ascaris suum (Xu et al. 2010; Chen et al. 2011; McCoy et al. 2015). Most of these studies used dsRNA as RNAi inducer in animal and human parasitic nematode in vitro and in vivo as well.

Table 2.

RNA interference done in various nematode parasites

SI.No.	Parasite	Target gene	Types of silencing RNA	Mode of introduction of RNA	Target effect	Authors
1	Ascaris suum	Enolase	dsRNA	Soaking	Reduction in parasite survival	Chen et al. (2011)
2	Haemonchus contortus		siRNA and dsRNA	Soaking Feeding and electroporation	Reduction of infectivity RNAi works only in limited genes	Geldhof et al. (2006), Naqvi et al. (2020)
3	Trichostrongylus colubriformis	Ubiquitin and Tropomyosin	dsRNA and siRNA	Soaking and Electroporation	Inhibition of larval development	Issa et al. (2005)
4	Ostertegia ostertegi	Tropomyosin, B-tubulin, ATP synthase, SOD, polyprotein allergen	dsRNA	Soaking and Electroporation	Target reduction can be achieved only in few genes	Visser et al. (2006)
5	Nippostrongylus braziliensis	Acetylcholin-esterase	dsRNA	Soaking	Target protein suppression	Hussein et al. (2002)
6	Onchocercus volvulus	Cathepsin L and Z-like cysteine proteases	dsRNA	Soaking	Inhibition of L3 molting	Lustigman et al. (2004), Ford et al. (2009)
7	Heligmosomoides polygyrus	Tropomysin	dsRNA	Feeding, electroporation and soaking	Study of gene function is limited due to deficiencies of genes involved in RNA- uptake and spread	Lendner et al. (2008)

In the case of H. contortus, there is a discrepancy in the target gene silencing via RNAi where some transcripts are not successfully silenced as target silencing is mainly achieved in those which are accessible to the environment such as parasites alimentary canal, excretory cell and nervous system like amphids (Samarasinghe 2010; Naqvi et al. 2020). It was observed that β-tubulin gene is consistently silenced where some gene failed to be silenced via RNAi in the case of H. contortus (Geldhof et al. 2006). Maule et al. (2011) concluded that RNAi could facilitate in vaccine-target validation in H. contortus as most of the vaccine candidates like H-11 or H-Gal-GP are environmentally exposed (Maule et al. 2011).

RNAi in cestode

RNAi has also been reported in few cestode parasites (Table 3). However, RNAi pathway works only on certain genes as in the case of nematodes; certain genes of Moniezia expansa are refractile to RNAi. Pierson et al. (2010) works on RNAi in M. expansa by soaking and electroporation targeting the neuropeptide F-gene (Me-npf-1) which causes transcriptional reduction with the phenotypic effect of tegumental aberration. Silencing of the targeted gene through RNAi was also reported in Echinococcus granulosus and E. multilocularis in which calmodulin gene was targeted in the former and 14-3-3 and elp genes in the latter by soaking and electroporation and observed a transcriptional as well as translational silencing of the genes (Mizukami et al. 2010; Mousavi et al. 2019).

Table 3.

RNA interference done in various cestode parasites

SI. no	Parasite	Target gene	Types of silencing RNA	Mode of introduce-tion of RNA	Target effect	Authors
1	Moniezia expansa	Neuropeptide F-gene (Me-npf-1)	dsRNA and siRNA	Soaking and Electropo-ration	Variable silencing of some gene only Tegumental aberration after silencing of actin gene	Pierson et al. (2010)
2	Echinococcus multilocularis	14-3-3 and elp genes	siRNA	-do-	Transcriptional and translational silencing	Mizukami et al. (2010)
3	E. granulosus	Calmodulin	dsRNA	-do-	Transcript suppression Phenotypic changes (lower viability, growth retardation, morphological abnormalities as well as EgCaM)	Mousavi et al. (2019)

Despite these promising results of RNAi in parasite functional studies, it has certain limitations and should be addressed sensibly for setting up RNAi as target validation platforms:

• (i)Optimization of parasite culture in vitro: in case of trematode parasite culture (F. gigantica), maintaining of the newly excysted juveniles (NEJs) in laboratory is difficult leading to unwanted termination of the experiment.
• (ii)Optimization of dsRNA/siRNA dose: an optimum dose of dsRNA is greatly influenced by mode of delivery of silencing RNA. For example, soaking method would usually require higher concentration of dsRNA in comparison to electroporation, feeding or through liposome. At the same time, an excessively higher dose could be detrimental to the parasite as it is for the case of electroporation.
• (iii)Transient efficacy of target silencing: RNA inducing gene silencing is not heritable to future generation unlike gene knock out through CRISPR-Cas9. It has been seen by many researchers that, reduction in the transcript of targeted gene is transient although, Dell’Oca et al. (2014) found that there is an amplification process of silencing signal in F. hepatica which is usually not seen in other helminth parasites. Temporary interference of target transcript likely reduces its utility in in vivo application of RNAi for parasitic control.

CRISPR-Cas9

In the past few decades, functional studies of parasite gene/protein have been carried out using RNAi, where there is target silencing of the transcript without heritable genetic manipulations or incorporation of a desired piece of DNA. CRISPR/Cas9 (Cluster Regularly Interspaced Short Palindromic Repeats/CRISPR Associated protein 9) is newer genetic engineering technique where the desired gene or a segment of DNA can be inserted into a genome or deleted at a precise location with high fidelity in prokaryotes as well as eukaryotes (Jinek et al. 2012; Doudna and Charpentier 2014). In short, this genome editing system utilized bacterial endonuclease, Cas9 to generate a double-strand break (DSB) at a locus of interest in the genome. The Cas9 is guided by guide RNA (gRNA) which is complementary to a sequence in the genome flanking a protospacer-adjacent motif (PAM) and the DNA strand break is thereby repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) (Fig. 2). Genome editing using CRISPR/Cas9 for functional studies has greatly advanced in protozoan parasites like Plasmodium falciparum (Ghorbal et al. 2014; Wagner et al. 2014), Toxoplasma gondii (Shen et al. 2014), Cryptosporidium parvum (Vinayak et al. 2015) Leishmania donovani (Zhang et al. 2017), Trypanosoma brucei (Rico et al. 2018) and Trichomonas vaginalis (Janssen et al. 2018).

Fig. 2.

Schematic diagram of CRISPR/Cas9 [Pictorial representation is designed by using online software (Biorender)]

The first adoption of this genome editing technique in helminth parasite was conducted on Strongyloides stercoralis and S. ratti where Gang et al. (2017) achieved disruption of twitchin gene unc-22 and subsequently via HDR which results in severe motility defects in adult and infective larva where the mutant parasites unlike unnoticeable phenotypic effect of RNAi in nematodes. Subsequently, researchers report standardizing CRISPR/Cas9 to edit the genomes of important flukes viz. Opisthorchis viverrini and Schistosoma mansoni (Arunsan et al. 2019; Ittiprasert et al. 2019). DNA double-strand break (DSBs) was directed in the gene coding for omega-1 ribonuclease of S. mansoni (Sm-omega-1 gene), speculated to causeTh2 polarization and granuloma formation and subsequent repaired through HDR. Upon injection of these mutant parasitic eggs in mice, there was failure to polarise Th2 response as well as reduced egg granuloma formation (Ittiprasert et al. 2019). In addition to genetic knock-out of a gene of omega-1 ribonuclease of S. mansoni, Sankaranarayanan et al. (2020) have achieved successful deletion of sulfo-transferase gene (SULT-OR) of S. mansoni (Sankaranarayanan et al. 2020).

In another report of gene knock-out in a fluke, O. viverrini, DSBs of granulin protein, thought to cause cancerous growth in the liver, encoded by Ov grn-1 gene was repaired through non-homologous end joining (NHEJ) where there is reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis (Arunsan et al. 2019). One of the disadvantages of RNAi which is temporary silencing of target transcript without heritable change which is addressed by CRISPR/Cas9 where genomic manipulation through INDEL is permanent as well as heritable (Gang et al. 2017).

Conclusion

Genetic manipulation via RNAi has opened up an avenue for functional studies in parasitic worms which otherwise is cumbersome and lengthy process. RNAi provides silencing of a target gene upon introduction of dsRNA complementary to the targeted site in parasite mRNA transcript. This robust tool has helped identification of key molecules for parasite invasion and parasite pathogenesis. Functional genomics study through CRIPSR/Cas9 offer improved opportunities as genetic alteration in terms of gene knock-out or knock-in of a desire piece of DNA forging heritable changes in parasites may be subjected for in vivo control of parasite. This novel approach of control by genetic manipulation yielding mutant parasite seems promising and worth exploring in human as well as livestock parasitism.

Author contributions

Both the authors contribute equally in preparation of this review manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

No additional data is generated other than which is listed in manuscript, table and figures. No additional data is generated other than which is listed in manuscript, table and figures.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

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