Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain

<em>Leishmania amazonensis</em> is one of the etiological agents of tegumentary and visceral leishmaniasis. Genome studies focusing on genes encoding virulence factors that play crucial roles in the establishment of the infection constitute an essential step towards the molecular characterization of a protozoan parasite that is being increasingly recognized as a significant human pathogen. Here, we report the sequencing and assembly of the <em>L</em><em>. amazonensis </em>PH8 strain combining data from long PacBio reads, short Illumina reads and synteny with the <em>Leishmania mexicana</em> genome. The final assembly, composed of 34 chromosomes, represents a genome of ~32 Mb with 8317 annotated genes. Several multigene families encoding virulence factors, such as A2, amastins, metalloproteins GP63 and cysteine proteases, were identified and compared to their annotation in the genomes of other <em>Leishmania</em> species. The <em>L. amazonensis </em>PH8 strain genome has 27 genes encoding all four sub-classes of amastins (α, β, γ and δ), 5 genes encoding A2 antigens, 9 genes encoding the metalloprotease GP63 and 76 genes encoding cysteine proteases. For all gene families analysed, we identified a larger number of copies and presented a more complete gene repertoire associated with <em>L. amazonensis </em>virulence factors, compared to previously published genomes. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we have also identified 14 genes encoding proteins involved in the parasite iron and heme metabolism and compared them to this gene repertoire in other Trypanosomatids. To follow these studies with a genetic approach that would allow us to directly address the role of these virulence factors, we tested two CRISPR-Cas9 protocols to generate <em>L. amazonensis</em> knockout cell lines. Using the Miltefosine Transporter (MT) gene as a proof of concept, we transfected promastigotes expressing the <em>Streptococcus pyogenes</em> Cas9 with <em>in vitro</em> transcribed sgRNA targeting this gene. As an alternative protocol, promastigotes were transfected with recombinant <em>Staphylococcus aureus </em>Cas9 complexed with sgRNAs. With both strategies, we were able to disrupt the MT gene with high efficiency, as shown by PCR and restriction digestion analysis as well as the development of miltefosine-resistant parasites.