First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites

Area of Science:

• Molecular Biology
• Parasitology
• Genetic Engineering

Background:

• Leishmania parasites, causative agents of leishmaniasis, are difficult to genetically modify using existing techniques.
• Efficient genome editing tools are crucial for understanding parasite biology and developing new treatments for leishmaniasis.

Purpose of the Study:

• To implement and validate the CRISPR-Cas9 system for efficient genome editing in Leishmania parasites.
• To demonstrate the utility of CRISPR-Cas9 for gene knockout, specifically targeting the paraflagellar rod-2 locus.

Main Methods:

• Expression of Cas9 endonuclease under the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter.
• Production of single-guide RNA under the U6snRNA promoter and terminator.
• CRISPR-Cas9 mediated knockout of the paraflagellar rod-2 gene family in a single transfection round.
• Whole genome sequencing to confirm the absence of off-target edits in generated null mutants.

Main Results:

• Efficient gene knockout of the paraflagellar rod-2 locus was achieved in Leishmania parasites.
• Null mutants were obtained in a single round of transfection, demonstrating high efficiency.
• Whole genome sequencing confirmed no off-target mutations in the selected clones.

Conclusions:

• The CRISPR-Cas9 system provides a powerful and efficient method for genome editing in Leishmania.
• This tool significantly surpasses previous genetic manipulation techniques for these protozoan pathogens.
• CRISPR-Cas9 opens new possibilities for functional genomics studies to accelerate leishmaniasis research.