Abstract
Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.
Highlights
Reverse genetics has been widely employed in studies to characterize the function of protein-coding genes
In Leishmania parasites, until recently, the function of a wide number of genes was assessed by gene disruption through homologous recombination (Cruz and Beverley, 1990), in which a recombination cassette containing a drug resistance marker and long sequences complementary to the target is delivered to the parasite where it is expected to replace the region of interest (ROI) in the genome (Cruz and Beverley, 1990)
To enable the integration of the pTB007 cassette in L. braziliensis M2903 for the stable expression of Cas9 and T7 RNA polymerase in this cell line, the L. major 5′ 344-bp and the 3′ 358-bp homology arms of pTB007 plasmid were replaced by a L. braziliensis conserved region of 322 bp in the 5′ and of 345 bp in the 3′ end of b-tubulin coding sequence by directional cloning (Figures 1A–C)
Summary
Reverse genetics has been widely employed in studies to characterize the function of protein-coding genes. The recombination usually occurs in one allele at a time and, more than one round of transfection, with different drug resistance markers, is needed in order to fully replace the ROI. This is an important limitation especially in Leishmania parasites in which aneuploidy is an event observed for different species and even strains of the same species (Rogers et al, 2011). A full knockout of a gene present in this chromosome requires four rounds of transfection with four different resistance markers (Rogers et al, 2011; Lachaud et al, 2014). Due to high genome plasticity in these organisms, other events such as gene duplication, amplification, alteration of ploidy states, and extrachromosomal elements are not rare, making the complete removal or substitution of a given gene especially challenging (Cruz et al, 1993; Rogers et al, 2011; Iantorno et al, 2017)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
