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

TY - JOUR

T1 - CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes

AU - Kovářová, Julie

AU - Novotná, Markéta

AU - Faria, Joana

AU - Rico, Eva

AU - Wallace, Catriona

AU - Zoltner, Martin

AU - Field, Mark C

AU - Horn, David

N1 - 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

PY - 2022/5

Y1 - 2022/5

N2 - 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.

AB - 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.

KW - CRISPR-Cas9

KW - Epigenetics

KW - Gene regulation

KW - Histone modification

KW - Trypanosoma brucei

UR - http://www.scopus.com/inward/record.url?scp=85127798454&partnerID=8YFLogxK

U2 - 10.1016/j.molbiopara.2022.111476

DO - 10.1016/j.molbiopara.2022.111476

M3 - Article

C2 - 35378143

SN - 0166-6851

VL - 249

JO - MOLECULAR AND BIOCHEMICAL PARASITOLOGY

JF - MOLECULAR AND BIOCHEMICAL PARASITOLOGY

M1 - 111476

ER -