CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Janssen, Brian D.; Chen, Yi-Pei; Molgora, Brenda M.; Wang, Shuqi E.; Simões-Barbosa, Augusto; Johnson, Patricia J.

doi:10.1038/s41598-017-18442-3

Cited by 46 publications

(36 citation statements)

References 75 publications

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“…However, when ssODN was provided, the DSBs generated by CRISPR/Cas9 editing were repairable by HDR, which is a different outcome to that observed with vertebrate cells where the HDR of the DSBs is extremely low compared to NHEJ, at least where both pathways are equally available [42]. In some single celled parasites including Plasmodium [43][44][45][46], Trichomonas vaginalis [47] and Cryptosporidium parvum [48], HDR is the only mechanism of DSB repair due to the lack of the NHEJ pathway; this leads to more accurate repair with less unintended or off-target effects. However, in Toxoplasma gondii, frequent NHEJ of DSBs occurs, resulting in random modifications [49,50].…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

You

Mayer

Johnston

et al. 2020

Preprint

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AbstractCRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. Here we report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. A CRISPR/Cas9-vector encoding gRNA X5 or X7 was introduced by electroporation into eggs recovered from livers of experimentally infected mice. Simultaneously, eggs were transfected with a ssODN donor encoding a stop codon in all six frames, flanked by 50 nt-long 5’- and 3’-homology arms matching the predicted Cas9-catalyzed double stranded break at X5 or X7. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programmed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, a significant decrease in circumoval granuloma size was observed in the lungs of the mice. Notably, there was an enhanced Th2 response involving IL-4, −5, −10, and-13 induced by lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, −13 and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.Author SummarySchistosomiasis is the most devastating of the parasitic helminth diseases. Currently, no vaccines are available for human use and praziquantel is the only available treatment raising considerable concern that drug resistance will develop. A major challenge faced by the schistosomiasis research community is the lack of suitable tools to effectively characterise schistosome gene products as potential new drug and/or vaccine targets. We introduced CRISPR/Cas9 mediated editing into S. mansoni eggs targeting the gene encoding acetylcholinesterase (AChE), a recognized anthelminthic drug target. We found that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). This platform provides a unique opportunity to generate precise loss-of-function insertions into the schistosome genome. We pre-screened the activity of two guide RNAs of the AChE gene and compared/validated the mutation efficacy using next-generation sequencing analysis at the genomic level and phenotypic modifications at the protein level. That resulted in reduced AChE activity observed in AChE-edited eggs, and decreased lung circumoval granuloma size in mice injected with those edited eggs. The CRISPR/Cas9-genome editing system we established in this study provides a pivotal platform for gene functional studies to identify and test new anti-schistosome intervention targets, which can be extended to the other human schistosome species and other important parasitic helminths.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

You

Mayer

Johnston

et al. 2020

Preprint

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“…Further even if such interactions do occur at a low frequency, it may be buffered by the presence of a higher percentage of ‘junk’ DNA than in procaryotes, therefore not interfering with DNA metabolism sufficiently to cause toxicity. Nevertheless, reports on definitive Cas9/dCas9 mediated toxicities in eucaryotes, especially single-celled organismssuch as Toxoplasma gondii [24], yeast [25], Trichomonas vaginilis [26], have begun to appear in literature. Plasmids carrying Cas9 have also shown toxicity in some cell lines where ribonucleoprotein delivery has improved viability [27].…”

Section: Discussionmentioning

confidence: 99%

Determination of Cas9/dCas9 associated toxicity in microbes

Misra

Bindal

Sodani

et al. 2019

Preprint

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23The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for 24 various applications. In case of the latter, a couple of previous studies had shown Cas9 protein 25 expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, 26 Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and 27 Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for 28 Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results 29 showed differential levels of Cas9 toxicity among the bacteria and lower transformation 30 efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated 31 lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate 32 Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, 33 Cas9/dCas9 associated toxicity was acute. 34 35 36 37 38 39 40 41 42 43 44 3 | P a g e Introduction: 45 Recombinant DNA technology together with High Throughput Sequencing in recent 46times, has allowed us to harvest a large amount of genetic information from the microbial 47 world. The technologies have been used extensively to find out which genes determine how 48 microbes, grow, travel, starve, cause diseases, ward of predators and even die. This information 49 is especially important for studying pathogenic bacteria, bacteria of industrial importance and 50 ones with special stress tolerance abilities. Perturbing the normal functioning of the genome 51 has emerged as the best method to probe function and dynamics of individual genes. 52 Discovery of the CRISPR -Cas viral defence systems opened up another novel and 53 efficient tool box for genome editing, gene silencing, targeted gene methylation, etc. in all 54 kinds of organisms from bacteria to humans [1][2][3][4]. The ease and efficiency of the system 55 has made it an extremely popular go-to system for various applications. The system has further 56 had widespread applications in metabolic engineering of bacteria as it allows easy 57 programming and multiplexing [5][6]. Among the CRISPR-Cas systems, the Cas9 system from 58 Streptomyces pyogens has gained popularity on account of being one of the earliest systems to 59 be discovered and its simplicity of usage [7][8][9]. The system comprises a single protein, Cas9 60 and the sgRNA , which together can be easily employed to bring about a host of desired changes 61 inside the cell of virtually any living being [10]. While the nucleoprotein, Cas9 itself has been 62 extensively used for genome editing [7][8][11][9], its nuclease deficient variant, dCas9 has 63 been useful for regulation of gene expression [3][1]. Systems employing the Cas9 variants have 64 shown great promise for use in eukaryotic, particularly mammalian systems [11]. Attempts to 65 use them in microbes have met with mixed success. 66 The Cas9/dCas9 and also Cas9 nickase systems were used successfully to probe gene 67 function...

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“…The supernatant was passed through a Vivaflow crossflow cassette using a 100-kDa molecular weight cutoff (MWCO) to separate the heavier exosomes (Exo > 100 kDa) from the lighter non-exosomal secreted fraction (NESF < 100 kDa). The level of NESF TvMIF was found to be 1.4-fold higher than that of Exo TvMIF by immunoblotting using an anti-TvMIF antibody ( 4 ) ( Fig. 2B ).…”

Section: Resultsmentioning

confidence: 97%

“…Thus, the mechanisms that drive parasite pathogenesis and the disease epidemiology are poorly understood ( 2 ). However, this is changing as more molecular and genetic tools for analyses are developed ( 3 , 4 ). The majority of T. vaginalis infections are asymptomatic.…”

Section: Introductionmentioning

confidence: 99%

Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival during Nutrient Stress

Chen

Twu

Johnson

2018

mBio

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Trichomonas vaginalis is responsible for the most prevalent non-viral sexually transmitted disease worldwide, and yet the mechanisms used by this parasite to establish and maintain infection are poorly understood. We previously identified a T. vaginalis homologue (TvMIF) of a human cytokine, human macrophage migration inhibitory factor (huMIF). TvMIF mimics huMIF’s role in increasing cell growth and inhibiting apoptosis in human host cells. To interrogate a role of TvMIF in parasite survival during infection, we asked whether overexpression of TvMIF (TvMIF-OE) confers an advantage to the parasite under nutrient stress conditions by comparing the survival of TvMIF-OE parasites to that of empty vector (EV) parasites. We found that under conditions of serum starvation, overexpression of TvMIF resulted in increased parasite survival. Serum-starved parasites secrete 2.5-fold more intrinsic TvMIF than unstarved parasites, stimulating autocrine and paracrine signaling. Similarly, we observed that addition of recombinant TvMIF increased the survival of the parasites in the absence of serum. Recombinant huMIF likewise increased the parasite survival in the absence of serum, indicating that the parasite may use this host survival factor to resist its own death. Moreover, TvMIF-OE parasites were found to undergo significantly less apoptosis and reactive oxygen species (ROS) generation under conditions of serum starvation, consistent with increased survival being the result of blocking ROS-induced apoptosis. These studies demonstrated that a parasitic MIF enhances survival under adverse conditions and defined TvMIF and huMIF as conserved survival factors that exhibit cross talk in host-pathogen interactions.

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CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis

Cited by 46 publications

References 75 publications

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

Determination of Cas9/dCas9 associated toxicity in microbes

Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival during Nutrient Stress

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