Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region...

Area of Science:

• Molecular Biology
• Parasitology
• Genetics

Background:

• Trypanosomatids, including Leishmania, are medically and economically significant eukaryotes with poorly understood biology.
• Leishmania exhibits high genomic plasticity, complicating genetic manipulation and the study of gene function.
• CRISPR-Cas9 has emerged as a valuable tool for investigating non-essential gene functions in these parasites.

Purpose of the Study:

• To develop a versatile, inducible gene editing system for Leishmania by combining CRISPR-Cas9 and Di-Cre recombinase.
• To create a molecular cloning-free, PCR-based method for efficient gene targeting.
• To overcome challenges associated with genomic plasticity in Leishmania.

Main Methods:

• Utilized a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase.
• Transfected donor DNAs and single-guide RNAs as PCR products.
• Integrated LoxP sequences using Cas9 and excised target genes via Cre-recombinase-mediated recombination, targeting intergenic regions to avoid protein interference.

Main Results:

• Developed a versatile system for inducible gene excision in Leishmania.
• Created 'universal' template plasmids for donor DNA cassettes, allowing flexible LoxP site placement.
• Demonstrated a fast, PCR-based, molecular cloning-free methodology.

Conclusions:

• The developed Di-Cre and CRISPR-Cas9 system is highly efficient and versatile for gene manipulation in Leishmania.
• This method effectively addresses the challenges posed by Leishmania's genomic plasticity.
• The system facilitates robust genetic studies of Leishmania by enabling precise gene targeting.