A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology.

We present a high-throughput CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna (T2A) to co-express a drug-selectable marker and a tagged protein from the same transcript. This dual-function design enables discrimination between transcriptional and post-translational regulation while maintaining native control elements.

We validate the system by tagging six Trypanosoma brucei proteins with fluorescent proteins and epitope tags and demonstrate: (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulated expression responding to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.