A high-throughput protein tagging toolkit that retains endogenous untranslated regions for studying gene regulation in kinetoplastids
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
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 CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses 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.
Date Issued
2025-02-26
Date Acceptance
2025-01-29
Citation
Open Biology, 2025, 15 (2)
ISSN
2046-2441
Publisher
The Royal Society
Journal / Book Title
Open Biology
Volume
15
Issue
2
Copyright Statement
© 2025 The Author(s). Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/39999874
Subjects
<italic>Trypanosoma brucei</italic>
3-UNTRANSLATED REGION
Biochemistry & Molecular Biology
CRISPR/Cas9
DEVELOPMENTAL REGULATION
endogenous tagging
EXPRESSION
gene regulation
GLUCOSE-TRANSPORTER
kinetoplastids
LEISHMANIA-MEXICANA
Life Sciences & Biomedicine
LOCATION
MESSENGER-RNA
POLYPROTEIN
PROCYCLIN
Science & Technology
T2A peptide
TRYPANOSOMA-BRUCEI
Publication Status
Published
Coverage Spatial
England
Article Number
240334
Date Publish Online
2025-02-26