Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

Area of Science:

• * Molecular Biology
• * Parasitology
• * Genomics

Background:

• * Leishmania parasites are crucial pathogens and model organisms, but functional genomics studies are hindered by low-throughput gene manipulation technologies.
• * Existing methods for gene disruption and tagging in Leishmania are limited, and RNA interference components are absent, restricting essential gene characterization.
• * The development of efficient genetic tools is vital for advancing research in Leishmania biology and pathogenesis.

Purpose of the Study:

• * To assess the efficacy of the T7 RNA polymerase-dependent CRISPR-Cas9 system for gene disruption and tagging in the model parasite Leishmania tarentolae.
• * To evaluate the performance of the glmS ribozyme-based system for inducible gene knock-down in Leishmania tarentolae.
• * To compare the capabilities and limitations of CRISPR-Cas9 and glmS systems for genetic manipulation in Leishmania.

Main Methods:

• * CRISPR-Cas9 system was employed for the targeted deletion of reference genes (Pf16, adenine phosphoribosyltransferase, Erv).
• * glmS ribozyme system was utilized for both constitutive and inducible gene knock-down studies using episomal and chromosomal reporters.
• * Leishmania tarentolae strains were cultivated in various media (BHI, MEM, IMDM, McCoy's 5A) to optimize inducible knock-down conditions.

Main Results:

• * Successful deletion of genes encoding flagellar motility factor Pf16 and adenine phosphoribosyltransferase was achieved using CRISPR-Cas9, yielding immotile and drug-resistant parasites, respectively.
• * CRISPR-Cas9 mediated gene disruption was unsuccessful for the mitochondrial flavoprotein Erv gene.
• * Constitutive down-regulation of an episomal mCherry-glmS reporter was observed (40-60%) in standard BHI medium.
• * Inducible knock-down of the episomal reporter exceeded 70% in supplemented MEM medium, dependent on glucosamine concentration.
• * Chromosomal glmS-tagging of Pf16, adenine phosphoribosyltransferase, or Erv genes did not result in a detectable knock-down phenotype.

Conclusions:

• * The CRISPR-Cas9 system is effective for gene disruption and tagging in Leishmania tarentolae, offering a valuable tool for functional genomics.
• * The glmS system demonstrated limitations, achieving only moderate efficiencies for episomal knock-downs and failing to produce a detectable phenotype for chromosomal knock-downs.
• * Further optimization or alternative strategies are needed to enhance the efficiency and applicability of the glmS system for chromosomal gene knock-down in Leishmania.