A DiCre recombinase-based system for inducible expression in Leishmania major

Abstract

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania.

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

“…A background cell line was established in which DiCre is expressed from the ribosomal locus, while both Cas9 and T7 polymerase are expressed from the tubulin array. For this, WT cells were transfected with plasmid pGL2339 [ 80 ], previously digested with Pac I and Pme I, to generate DiCre-expressing cells. Correct integration into the ribosomal locus was confirmed by PCR.…”

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9–1–1 complex in the maintenance of the plastic genome ofLeishmania

“…The DiCre (pGL2339 (20)) and HUS1 Flox -expressing constructs were generated by gateway recombination reactions. For the DiCre construct, a BP reaction was performed to introduce the 5′SSU (253 bp) and 3′SSU (955 bp) homology sequences into pDONR P41-Pr and pDONR P2rP3 vectors, respectively.…”

“…We have previously shown that HUS1 is involved in the Leishmania major response to genotoxic stress (17,18), but the roles that are critical for the parasite’s survival have not been determined. In this study, we have adapted the DiCre-mediated gene deletion system (19,20) to be used in L. major and reveal the essentiality of HUS1. We have advanced our understanding of HUS1 function at the G2/M checkpoint by demonstrating that its absence leads to aberrant mitosis onset in the presence of DNA damage in both unperturbed and replication-stressed cells.…”

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

“…The repair of the double strand breaks (DSB) at the 5′ and 3′ ends of the GOI allows the integration of the LoxP sequences and resistance cassettes to generate a Floxed targeted gene cell line (Figure 1c). These edited parasites are obtained after a single round of transfection, which generally takes 8–10 days, making this method highly efficient and time‐saving compared with the previously published conditional KO method (Santos et al, ). In some cases, most probably because certain genes cannot tolerate untranscribed region (UTR) modifications, the strategy in which the GOI is directly flanked by LoxP sequences showed limited efficiency; and edited parasites with modified UTRs did not recover after transfection or exhibited growth defects and/or phenotype changes before induction.…”