Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

Abstract

Leishmania parasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited in Leishmania because of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Beneke et al. and the glmS ribozyme-based knockdown system in the model parasite Leishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in alternative media and identified supplemented MEM, IMDM and McCoy's 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentrationdependent down-regulation of the episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv

“…While mixed disulfides between Sc Mia40 and Sc Erv1 or incoming substrates are long-lived and detectable by Western blot analysis ( 18 , 48 , 53 ), Lt Erv seems to form short-lived mixed disulfide bonds that are difficult to detect. The addition of small peptide tags to the N or C terminus of Lt Erv did not impair parasite growth, in contrast to previous attempts to tag Lt Erv with bulky mCherry ( 33 ). Parasites with an altered shuttle pair CQVYC-motif to slow down dithiol-disulfide exchange reactions were not viable.…”

“…To purify interaction partners by affinity chromatography and to gain insights into the redox interactome of Lt Erv, we generated L. tarentolae cell lines that chromosomally encode Lt Erv with a C-terminal His 8 -tag. Using the CRISPR-Cas9 system ( 33 ), we introduced a chromosomal double-strand break downstream of the LTERV open reading frame and provided a DNA repair fragment with homology regions that flank the tag-coding sequence and a resistance cassette against puromycin (see Fig. S1a in the supplemental material).…”

“…To perform quantitative mass spectrometry, we established a protocol for SILAC (stable isotope labeling by amino acids in cell culture). Previous attempts to find a suitable medium for L. tarentolae without fetal bovine serum (FBS) (as a confounding source for arginine or lysine) or with dialyzed FBS had failed ( 33 ). We therefore tested five alternative medium compositions based on M199, which had been used in SILAC experiments with Leishmania donovani ( 35 ) (Fig.…”

“…The Erv-encoding genes in Leishmania infantum and Leishmania infantum tarentolae are essential ( 32 , 33 ), and endogenous and heterologous Lt Erv was shown to be imported into the IMS in L. tarentolae and in yeast, respectively ( 2 , 28 ). Furthermore, RNAi knockdowns of Tb Erv1 caused a growth arrest ( 27 ), impaired the mitochondrial protein import of 13 proteins with twin Cx 3,9 C motifs, and led to the identification of 25 candidate substrates in the IMS of T. brucei ( 24 ).…”

Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

“…To introduce mutations in recodonized PFKELCH13, the gene was first subcloned into plasmid pET45b using restriction sites AvrII and XhoI, followed by site-directed mutagenesis with primers P71 -P76 or P84 -P87, and recloning into pSLI-N-GFP-2xFKBP-loxP(K13) using primers P65 and P69. Primers P88 and P89 were used to amplify the glmS and M9 sequences from plasmids pCR 2.1 TOPO-mCherry-glmS and pCR 2.1 TOPO-mCherry-M9, respectively 46 . The loxP site from pSLI-His8-PFKELCH13 was subsequently excised and replaced with the glmS or M9 sequence using StuI and XhoI, yielding pSLI-His8-PFKELCH13-glmS and pSLI-His8-PFKELCH13-M9.…”