A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Kinetoplastids are significant pathogens requiring advanced genetic manipulation tools.
• Current methods for generating kinetoplastid mutants are often time-consuming and labor-intensive.
• CRISPR-Cas9 technology offers a promising avenue for efficient genetic engineering.

Purpose of the Study:

• To develop a user-friendly CRISPR-Cas9 toolkit for rapid gene tagging and knockout in kinetoplastids.
• To create scalable methods for high-throughput generation of mutant kinetoplastid phenotypes.
• To facilitate functional studies and localization screening of kinetoplastid proteins.

Main Methods:

• Development of a CRISPR-Cas9 toolkit requiring no DNA cloning.
• Implementation of single-guide RNA delivery via PCR-generated DNA templates and T7 RNA polymerase.
• Utilizing an online resource (LeishGEdit.net) for automated primer design.
• Creation of plasmids for rapid DNA construct generation and transfection.

Main Results:

• Successful knock-in of various tags (fluorescent proteins, BirA*, luciferase, HaloTag, epitope tags) at N- or C-termini.
• Generation of null mutants in *Leishmania major*, *Leishmania mexicana*, and *Trypanosoma brucei* in a single transfection round.
• Undetectable deleted genes in non-clonal populations, demonstrating high efficiency.
• Establishment of rapid and large-scale knockout screening capabilities.

Conclusions:

• The developed CRISPR-Cas9 toolkit significantly accelerates genetic manipulation in kinetoplastids.
• This technology enables high-throughput functional genomics and drug target validation in these pathogens.
• The toolkit provides a scalable and efficient platform for diverse kinetoplastid species.