LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella.

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania.

The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat.

Eukaryotic flagella undertake different beat types as necessary for different functions; for example, the Leishmania parasite flagellum undergoes a symmetric tip-to-base beat for forward swimming and an asymmetric base-to-tip beat to rotate the cell. In multi-ciliated tissues or organisms, the asymmetric beats are coordinated, leading to movement of the cell, organism or surrounding fluid. This coordination involves a polarisation of power stroke direction. Here, we asked whether the asymmetric beat of the single Leishmania flagellum also has a fixed polarisation. We developed high frame rate dual-colour fluorescence microscopy to visualise flagellar-associated structures in live swimming cells. This showed that the asymmetric Leishmania beat is polarised, with power strokes only occurring in one direction relative to the asymmetric flagellar machinery. Polarisation of bending was retained in deletion mutants whose flagella cannot beat but have a static bend. Furthermore, deletion mutants for proteins required for asymmetric extra-axonemal and rootlet-like flagellum-associated structures also retained normal polarisation. Leishmania beat polarisation therefore likely arises from either the nine-fold rotational symmetry of the axoneme structure or is due to differences between the outer doublet decorations.

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi.

Cardiolipin depletion-induced changes in the Trypanosoma brucei proteome.

The mitochondrial signature glycerophospholipid, cardiolipin (CL), binds to transporters of the inner mitochondrial membrane and plays a central role in formation and stability of respiratory supercomplexes. Functional and structural requirement of CL for mitochondrial membrane proteins has been studied in vitro using purified reconstituted proteins or in CL synthesis knockout cells that are viable under specific growth conditions. However, no information is available on mitochondrial function, protein stability, or expression levels in cells during CL depletion. In contrast to yeast and mammalian cells, CL synthesis is essential in Trypanosoma brucei. By stable isotope labeling with amino acids in cell culture and mass spectrometry, we analyzed protein levels in T. brucei procyclic forms at different time points during depletion of CL using tightly controllable conditional CL synthase knockout mutants and identified a set of novel CL-dependent proteins (CLDPs) with unknown functions. Depletion of individual CLDPs using knockout or knockdown technologies showed that although CL synthesis is essential, expression of a given CLDP is not. In addition, ablation of CL synthesis leads to respiratory supercomplex instability and altered mitochondrial ultrastructure and function. Our findings suggest that CL may bind to and affect many more proteins in eukaryotes than previously thought.-Schädeli, D., Serricchio, M., Ben Hamidane, H., Loffreda, A., Hemphill, A., Beneke, T., Gluenz, E., Graumann, J., Bütikofer, P. Cardiolipin depletion-induced changes in the Trypanosoma brucei proteome.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

Comparative Life Cycle Transcriptomics Revises Leishmania mexicana Genome Annotation and Links a Chromosome Duplication with Parasitism of Vertebrates.

Leishmania spp. are protozoan parasites that have two principal life cycle stages: the motile promastigote forms that live in the alimentary tract of the sandfly and the amastigote forms, which are adapted to survive and replicate in the harsh conditions of the phagolysosome of mammalian macrophages. Here, we used Illumina sequencing of poly-A selected RNA to characterise and compare the transcriptomes of L. mexicana promastigotes, axenic amastigotes and intracellular amastigotes. These data allowed the production of the first transcriptome evidence-based annotation of gene models for this species, including genome-wide mapping of trans-splice sites and poly-A addition sites. The revised genome annotation encompassed 9,169 protein-coding genes including 936 novel genes as well as modifications to previously existing gene models. Comparative analysis of gene expression across promastigote and amastigote forms revealed that 3,832 genes are differentially expressed between promastigotes and intracellular amastigotes. A large proportion of genes that were downregulated during differentiation to amastigotes were associated with the function of the motile flagellum. In contrast, those genes that were upregulated included cell surface proteins, transporters, peptidases and many uncharacterized genes, including 293 of the 936 novel genes. Genome-wide distribution analysis of the differentially expressed genes revealed that the tetraploid chromosome 30 is highly enriched for genes that were upregulated in amastigotes, providing the first evidence of a link between this whole chromosome duplication event and adaptation to the vertebrate host in this group. Peptide evidence for 42 proteins encoded by novel transcripts supports the idea of an as yet uncharacterised set of small proteins in Leishmania spp. with possible implications for host-pathogen interactions.

Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1.

In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock-down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.

Histone H1 plays a role in heterochromatin formation and VSG expression site silencing in Trypanosoma brucei.

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6-8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis.

BACKGROUND: Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method. RESULTS: We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically. CONCLUSIONS: Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum.

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology.

The cell cycle is central to understanding fundamental biology of Leishmania, a group of human-infective protozoan parasites. Leishmania have two main life cycle morphologies: the intracellular amastigote in the mammalian host and the promastigote in the fly. We have produced the first comprehensive and quantitative description of a Leishmania promastigote cell cycle taking a morphometric approach to position any cell within the cell cycle based on its length and DNA content. We describe timings of cell cycle phases and rates of morphological changes; kinetoplast and nucleus S phase, division and position, cell body growth and morphology changes, flagellum growth and basal body duplication. We have shown that Leishmania mexicana undergoes large changes in morphology through the cell cycle and that the wide range of morphologies present in cultures during exponential growth represent different cell cycle stages. We also show promastigote flagellum growth occurs over multiple cell cycles. There are clear implications for the mechanisms of flagellum length regulation, life cycle stage differentiation and trypanosomatid division in general. This data set therefore provides a platform which will be of use for post-genomic analyses of Leishmania cell biology in relation to differentiation and infection.

Depletion of mitochondrial acyl carrier protein in bloodstream-form Trypanosoma brucei causes a kinetoplast segregation defect.

Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.

Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase.

We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.

Beyond 9+0: noncanonical axoneme structures characterize sensory cilia from protists to humans.

The intracellular amastigote stages of parasites such as Leishmania are often referred to as aflagellate. They do, however, possess a short axoneme of cryptic function. Here, our examination of the structure of this axoneme leads to a testable hypothesis of its role in the cell biology of pathogenicity. We show a striking similarity between the microtubule axoneme structure of the Leishmania mexicana parasite infecting a macrophage and vertebrate primary cilia. In both, the 9-fold microtubule doublet symmetry is broken by the incursion of one or more microtubule doublets into the axoneme core, giving rise to an architecture that we term here the 9v (variable) axoneme. Three-dimensional reconstructions revealed that no particular doublet initiated the symmetry break, and moreover it often involved 2 doublets. The tip of the L. mexicana flagellum was frequently intimately associated with the macrophage vacuole membrane. We propose that the main function of the amastigote flagellum is to act as a sensory organelle with important functions in host-parasite interactions and signaling in the intracellular stage of the L. mexicana life cycle.

Ros3 (Lem3p/CDC50) Gene Dosage Is Implicated in Miltefosine Susceptibility in Leishmania (Viannia) braziliensis Clinical Isolates and in Leishmania (Leishmania) major.

The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the ß-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis ß-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major ß-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) ß-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.

Bar-seq strategies for the LeishGEdit toolbox.

The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we previously developed a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligo sequences are now accessible through our website www.leishgedit.net allowing researchers to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.