Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11110%2F22%3A10444028" target="_blank" >RIV/00216208:11110/22:10444028 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/22:10444028

Výsledek na webu

<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=ijalpYZUCg" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=ijalpYZUCg</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1098/rsob.210361" target="_blank" >10.1098/rsob.210361</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

Popis výsledku v původním jazyce

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Název v anglickém jazyce

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

Popis výsledku anglicky

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2022

Kód důvěrnosti údajů

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

Název periodika

Open Biology

ISSN

2046-2441

e-ISSN

2046-2441

Svazek periodika

12

Číslo periodika v rámci svazku

4

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

14

Strana od-do

210361

Kód UT WoS článku

000792699400002

EID výsledku v databázi Scopus

2-s2.0-85128969366