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

0498707 - BC 2019 RIV US eng J - Journal Article
Ishemgulova, Aygul - Hlaváčová, J. - Majerová, K. - Butenko, Anzhelika - Lukeš, Julius - Votýpka, Jan - Volf, P. - Yurchenko, Vyacheslav
CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1.
PLoS ONE. Roč. 13, č. 2 (2018), č. článku e0192723. ISSN 1932-6203. E-ISSN 1932-6203
R&D Projects: GA ČR(CZ) GA16-18699S; GA MŠMT(CZ) LL1601
Grant - others:Grantová agentura České republiky (GA ČR)(CZ) GA17-10656S; EU(XE) COST Action CM1307
Institutional support: RVO:60077344
Keywords : BATTEN-DISEASE * TRYPANOSOMA-BRUCEI * EXPRESSION SYSTEM * GENOME * GLN3 * TRANSMISSION * PHYLOGENIES * TARENTOLAE * INSIGHTS
OECD category: Reproductive biology (medical aspects to be 3)
Impact factor: 2.776, year: 2018
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192723

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.
Permanent Link: http://hdl.handle.net/11104/0290989