CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes

Julie Kovářová, Markéta Novotná, Joana Faria, Eva Rico, Catriona Wallace, Martin Zoltner, Mark C. Field, David Horn (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)
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Abstract

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

Original languageEnglish
Article number111476
Number of pages7
JournalMolecular and Biochemical Parasitology
Volume249
Early online date1 Apr 2022
DOIs
Publication statusPublished - 10 Apr 2022

Keywords

  • CRISPR-Cas9
  • Epigenetics
  • Gene regulation
  • Histone modification
  • Trypanosoma brucei

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