CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes
The result's identifiers
Result code in 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>
Alternative codes found
RIV/00216208:11310/22:10449796
Result on the web
<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>
Alternative languages
Result language
angličtina
Original language name
CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes
Original language description
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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology
Result continuities
Project
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Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2022
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Molecular and Biochemical Parasitology
ISSN
0166-6851
e-ISSN
1872-9428
Volume of the periodical
249
Issue of the periodical within the volume
MAY
Country of publishing house
NL - THE KINGDOM OF THE NETHERLANDS
Number of pages
7
Pages from-to
111476
UT code for WoS article
000913149200002
EID of the result in the Scopus database
2-s2.0-85127798454