Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

Gene editing in trypanosomatids has long been proven difficult. The development of CRISPR-Cas9 has improved this issue, opening the way to a better understanding of biological processes and drug resistance mechanisms, and screening of drug targets. Different strategies have now been developed: either PCR-or plasmid-based, mainly differing by the nature of the donor DNA and the single guide RNA transcription. Here we review the main genetic tools available in Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei for gene tagging, single base editing and deletion of non-essential and essential genes. We discuss the main advantages and challenges of different strategies and how to choose 'the right cut' depending on the importance of untranslated regions. These considerations allow selection of the most accurate gene editing approach for a given functional analysis.

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA) might have important evolutionary and functional implications, but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two Leishmania clonal populations representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations. MA usually affected a defined subset of chromosomes, of which some display enrichment in snoRNA genes which could represent an adaptative benefit to the amplification of these chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

14Maintenance of stable ploidy over continuous mitotic events is a paradigm for most higher 15 eukaryotes. Defects in chromosome segregation and/or replication can lead to aneuploidy, a 16 condition often considered deleterious. However, in Leishmania, a Protozoan parasite, 17aneuploidy is a constitutive feature, where variations of somies represent a mechanism of gene 18 expression adaptation, possibly impacting phenotypes. Strikingly, clonal Leishmania 19 populations display cell-to-cell somy variation, a phenomenon named mosaic aneuploidy (MA). 20However, until recently, no method was available for the determination of the complete 21 karyotype of single Leishmania parasites. To overcome this limitation, we used here for the first 22 time a high-throughput single-cell genomic sequencing (SCGS) method to estimate individual 23 karyotypes of 1560 promastigote cells in a clonal population of Leishmania donovani. We 24 identified 128 different karyotypes, of which 4 were dominant. A network analysis revealed that 25 most karyotypes are linked to each other by changes in copy number of a single chromosome 26 and allowed us to propose a hypothesis of MA evolution. Moreover, aneuploidy patterns that 27 were previously described by Bulk Genome Sequencing as emerging during first contact of 28 promastigotes populations with different drugs are already pre-existing in single karyotypes in 29 the SCGS data, suggesting a (pre-)adaptive role of MA. Additionally, the degree of somy 30 variation was chromosome-specific. The SCGS also revealed a small fraction of cells where one 31 or more chromosomes were nullisomic. Together, these results demonstrate the power of SCGS 32to resolve sub-clonal karyotype heterogeneity in Leishmania and pave the way for 33understanding the role of MA in these parasites' adaptability. 34 35