scholarly journals CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Greg Matlashewski
Keyword(s):  
Visceral Leishmaniasis ◽  
Genome Editing ◽  
Cleavage Site ◽  
Leishmania Donovani ◽  
Expression Vectors ◽  
Drug Selection ◽  
End Joining ◽  
Content Type ◽  
Homology Directed Repair ◽  
Cas9 Nuclease

ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. IMPORTANCE Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Development of a Multiplexed Assay for Detection ofLeishmania donovaniandLeishmania infantumProtein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Claudia Abeijon ◽  
Fabiana Alves ◽  
Severine Monnerat ◽  
Monique Wasunna ◽  
Jane Mbui ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Diagnostic Test ◽  
Leishmania Donovani ◽  
Protein Biomarkers ◽  
Serological Tests ◽  
Assay Sensitivity ◽  
Content Type ◽  
Detection Assay ◽  
Previously Treated ◽  
New Biomarkers

ABSTRACTVisceral leishmaniasis (VL) is a serious and fatal disease caused by the parasitesLeishmania infantumandLeishmania donovani. The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified threeL. infantumprotein biomarkers (Li-isd1,Li-txn1, andLi-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused byL. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused byL. donovani. Here, we report the discovery and characterization of two new biomarkers ofL. donovani(Ld-mao1andLd-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection ofLd-mao1andLd-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

scholarly journals Microhomologies are prevalent at Cas9-induced larger deletions

2019 ◽  
Vol 47 (14) ◽  
pp. 7402-7417 ◽  
Author(s):  
Dominic D G Owens ◽  
Adam Caulder ◽  
Vincent Frontera ◽  
Joe R Harman ◽  
Alasdair J Allan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Biomedical Research ◽  
High Frequency ◽  
Cultured Cells ◽  
Mouse Embryos ◽  
Deletion Breakpoint ◽  
End Joining ◽  
Crispr System ◽  
Cas9 Nuclease ◽  
Target Sites

Abstract The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

scholarly journals CpG Oligodeoxynucleotide 2006 and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis

2011 ◽  
Vol 55 (7) ◽  
pp. 3461-3464 ◽  
Author(s):  
Suman Gupta ◽  
Shraddha A. Sane ◽  
Nishi Shakya ◽  
Preeti Vishwakarma ◽  
W. Haq
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Inhibitory Effect ◽  
Rational Approach ◽  
Host Use ◽  
Cpg Odn ◽  
Content Type ◽  
Cpg Oligodeoxynucleotide ◽  
Class B ◽  
Different Doses

ABSTRACTIn view of the severe immunosuppression in visceral leishmaniasis (VL), a rational approach to effectively combat the parasitic scourge would be to enhance the immune status of the host. Use of CpG oligodeoxynucleotide (CpG-ODN) against leishmaniasis has previously been reported, especially as an immunomodulator and adjuvant with various immunogens. In the present study, experiments were carried out with BALB/c mice and hamsters infected withLeishmania donovani. Immunostimulating class B bacterial CpG-ODN namely, ODN-2006, was administered at various doses by the intraperitoneal (i.p.) route. The dose of CpG-ODN-2006 (1 nM/single dose) showing the most antileishmanial activity was given as free and liposomal forms with different doses of miltefosine, namely, 5 and 10 mg/kg of body weight, for 5 days in mice and hamsters, respectively. Among the various groups, mice coadministered liposomal CpG-ODN and miltefosine (5 mg/kg) showed the best inhibitory effect (97% parasite inhibition) compared with free CpG-ODN plus miltefosine and miltefosine, free CpG-ODN, and liposomal CpG-ODN given separately. Similar responses were observed in the case of hamsters, where the combination of liposomal CpG-ODN with miltefosine (10 mg/kg) gave 96% parasite inhibition. Promising antileishmanial efficacy was observed in animals treated with liposomal CpG-ODN and miltefosine.

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

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

2019 ◽  
Author(s):  
Tien Van Vu ◽  
Velu Sivankalyani ◽  
Eun-Jung Kim ◽  
Duong Thi Hai Doan ◽  
Mil Thi Tran ◽  
...  
Keyword(s):  
Genome Editing ◽  
De Novo ◽  
Phenotypic Selection ◽  
Culture Conditions ◽  
Nonhomologous End Joining ◽  
Physical Culture ◽  
End Joining ◽  
Dark Cycle ◽  
Homology Directed Repair ◽  
Error Prone Repair

ABSTRACTGenome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31°C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mM NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually as well as sexually reproducing plants.

scholarly journals A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei-Yi Yan ◽  
Si-Shang Li ◽  
Xin-Yuan Ding ◽  
Xiao-Peng Guo ◽  
Qi Jin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Editing ◽  
Large Scale ◽  
Nonhomologous End Joining ◽  
Genetic Mutations ◽  
Repair Pathway ◽  
End Joining ◽  
Content Type ◽  
Nhej Repair ◽  
Short Palindromic Repeat

ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

scholarly journals Leishmania donovani Lipophosphoglycan Increases Macrophage-Dependent Chemotaxis of CXCR6-Expressing Cells via CXCL16 Induction

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Visnu Chaparro ◽  
Louis-Philippe Leroux ◽  
Aude Zimmermann ◽  
Armando Jardim ◽  
Brent Johnston ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Molecular Mechanisms ◽  
Defense Mechanism ◽  
Protozoan Parasite ◽  
Parasitic Infections ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Content Type

ABSTRACT CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro. Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.

scholarly journals Nanotized Curcumin and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Brajendra Tiwari ◽  
Richa Pahuja ◽  
Pradeep Kumar ◽  
Srikanta Kumar Rath ◽  
Kailash Chand Gupta ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Oral Drug ◽  
Hamster Model ◽  
Content Type ◽  
Leishmanicidal Activity ◽  
Traditional Indian Medicine ◽  
Indian Medicine

ABSTRACT Leishmaniasis chemotherapy remains very challenging due to high cost of the drug and its associated toxicity and drug resistance, which develops over a period of time. Combination therapies (CT) are now in use to treat many diseases, such as cancer and malaria, since it is more effective and affordable than monotherapy. CT are believed to represent a new explorable strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. In the present study, we investigated the effect of a combination of a traditional Indian medicine (ayurveda), a natural product curcumin and miltefosine, the only oral drug for visceral leishmaniasis (VL) using a Leishmania donovani-hamster model. We developed an oral nanoparticle-based formulation of curcumin. Nanoformulation of curcumin alone exhibited significant leishmanicidal activity both in vitro and in vivo. In combination with miltefosine, it exhibited a synergistic effect on both promastigotes and amastigotes under in vitro conditions. The combination of these two agents also demonstrated increased in vivo leishmanicidal activity accompanied by increased production of toxic reactive oxygen/nitrogen metabolites and enhanced phagocytic activity. The combination also exhibited increased lymphocyte proliferation. The present study thus establishes the possible use of nanocurcumin as an adjunct to antileishmanial chemotherapy.

scholarly journals Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
April C. Joice ◽  
Sihyung Yang ◽  
Abdelbasset A. Farahat ◽  
Heidi Meeds ◽  
Mei Feng ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Treatment Strategies ◽  
Parasite Burden ◽  
Pharmacokinetic Analysis ◽  
Content Type ◽  
New Treatment ◽  
Almost All

ABSTRACT Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

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