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CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

Identifikátory výsledku

  • Kód výsledku v IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60076658%3A12310%2F18%3A43897402" target="_blank" >RIV/60076658:12310/18:43897402 - isvavai.cz</a>

  • Nalezeny alternativní kódy

    RIV/60077344:_____/18:00498707 RIV/61988987:17310/18:A1901WOT RIV/00216208:11310/18:10373352

  • Výsledek na webu

    <a href="https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0192723&type=printable" target="_blank" >https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0192723&type=printable</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.1371/journal.pone.0192723" target="_blank" >10.1371/journal.pone.0192723</a>

Alternativní jazyky

  • Jazyk výsledku

    angličtina

  • Název v původním jazyce

    CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

  • Popis výsledku v původním jazyce

    Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM. 22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM. 22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM. 22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM. 22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.

  • Název v anglickém jazyce

    CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

  • Popis výsledku anglicky

    Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM. 22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM. 22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM. 22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM. 22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.

Klasifikace

  • Druh

    J<sub>imp</sub> - Článek v periodiku v databázi Web of Science

  • CEP obor

  • OECD FORD obor

    10608 - Biochemistry and molecular biology

Návaznosti výsledku

  • Projekt

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

  • Návaznosti

    I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Ostatní

  • Rok uplatnění

    2018

  • Kód důvěrnosti údajů

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

Údaje specifické pro druh výsledku

  • Název periodika

    PLoS One

  • ISSN

    1932-6203

  • e-ISSN

  • Svazek periodika

    13

  • Číslo periodika v rámci svazku

    2

  • Stát vydavatele periodika

    US - Spojené státy americké

  • Počet stran výsledku

    17

  • Strana od-do

  • Kód UT WoS článku

    000425083400048

  • EID výsledku v databázi Scopus

    2-s2.0-85042073182