A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• CRISPR/Cas9 is a powerful tool for gene editing in Trypanosoma brucei.
• Current methods require stable expression of Cas9 and T7 RNA polymerase (T7RNAP) in limited cell lines.

Purpose of the Study:

• To develop a versatile CRISPR/Cas9 system for genome editing in any Trypanosoma brucei cell line.
• To enable transient expression of necessary editing components.

Main Methods:

• A sequential transfection expression system was developed.
• Transient expression of Cas9 and T7RNAP was achieved.
• PCR products for guide RNAs (gRNAs) and repair templates were delivered.

Main Results:

• The system allows for genome editing without stable integration of Cas9 and T7RNAP genes.
• This method is applicable to any Trypanosoma brucei cell line.

Conclusions:

• This novel system significantly expands the utility of CRISPR/Cas9 for Trypanosoma brucei research.
• It facilitates gene knockouts and in situ tagging across diverse cell lines.