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A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

ABSTRACT: Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

SUBMITTER: Lypaczewski P

PROVIDER: S-EPMC6224596 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Lypaczewski Patrick P Hoshizaki Johanna J Zhang Wen-Wei WW McCall Laura-Isobel LI Torcivia-Rodriguez John J Simonyan Vahan V Kaur Amanpreet A Dewar Ken K Matlashewski Greg G

Scientific reports 20181108 1

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third ...[more]

PMID: 30409989

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Project description:UnlabelledThe 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.ImportanceLeishmania 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.

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A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Publications

A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

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