Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in...

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• CRISPR-Cas9 is a powerful tool for genome editing in trypanosomatids like Trypanosoma and Leishmania.
• Traditional CRISPR/Cas9 methods in these parasites have limitations including reliance on specific strains, selectable markers, and potential genomic instability.
• These limitations hinder broad applicability across diverse parasite strains and complex genomic modifications.

Purpose of the Study:

• To develop and optimize a marker-free CRISPR-Cas9 genome editing method for trypanosomatid parasites.
• To overcome the drawbacks associated with conventional CRISPR/Cas9 strategies in these organisms.
• To provide a flexible and efficient platform for functional genomics in kinetoplastids.

Main Methods:

• Transient ribonucleoprotein (RNP) complex delivery via electroporation.
• Design of guide RNA (gRNA) and repair templates (cassettes).
• Marker-free protocol involving RNP assembly, electroporation, and PCR/sequencing for clonal screening.

Main Results:

• Achieved high genome editing efficiency without plasmid integration or antibiotic selection.
• Enabled rapid (≤3 weeks) generation of homozygous mutant lines in wild-type strains.
• Demonstrated suitability for multiplexed editing in polyploid genomes and validation of essential genes.

Conclusions:

• The optimized marker-free CRISPR-Cas9 RNP delivery method significantly improves genome editing in trypanosomatids.
• This technique offers a reproducible, efficient, and adaptable solution for genetic manipulation in diverse kinetoplastid species.
• The workflow facilitates rapid functional genomics studies, including multiplexed gene editing and essential gene validation.