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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F22%3A00569165" target="_blank" >RIV/60077344:_____/22:00569165 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/22:10449796

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0166685122000305?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0166685122000305?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.molbiopara.2022.111476" target="_blank" >10.1016/j.molbiopara.2022.111476</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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 immunofluorescencebased localisation and for enriching protein complexes, (GFP)HAT2 or (GFP)HDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

Název v anglickém jazyce

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

Popis výsledku anglicky

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 immunofluorescencebased localisation and for enriching protein complexes, (GFP)HAT2 or (GFP)HDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Molecular and Biochemical Parasitology

ISSN

0166-6851

e-ISSN

1872-9428

Svazek periodika

249

Číslo periodika v rámci svazku

MAY

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

7

Strana od-do

111476

Kód UT WoS článku

000913149200002

EID výsledku v databázi Scopus

2-s2.0-85127798454