Kovářová, Julie, Novotná, Markéta, Faria, Joana orcid.org/0000-0001-6274-8143 et al. (5 more authors) (2022) CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes. MOLECULAR AND BIOCHEMICAL PARASITOLOGY. 111476. ISSN 0166-6851
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.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | Funding Information: This work was supported by an Investigator Award to D.H. (217105/Z/19/Z) and a Centre Award (203134/Z/16/Z) from the Wellcome Trust . M.N. was supported by a four-Year PhD Studentship (222326/Z/21/Z) from the Wellcome Trust. The FingerPrints Proteomics Facility is supported by the 'Wellcome Trust Technology Platform' award (097945/B/11/Z). Publisher Copyright: © 2022 The Authors |
Keywords: | CRISPR-Cas9,Epigenetics,Gene regulation,Histone modification,Trypanosoma brucei |
Dates: |
|
Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 12 Apr 2022 10:40 |
Last Modified: | 21 Jan 2025 18:02 |
Published Version: | https://doi.org/10.1016/j.molbiopara.2022.111476 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1016/j.molbiopara.2022.111476 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:185652 |
Download
Filename: 1_s2.0_S0166685122000305_main.pdf
Description: 1-s2.0-S0166685122000305-main
Licence: CC-BY 2.5