Author(s):
Espada, Caroline R. ; Quilles, José Carlos ; Albuquerque-Wendt, Andreia ; Cruz, Mario C. ; Beneke, Tom ; Lorenzon, Lucas B. ; Gluenz, Eva ; Cruz, Angela K. ; Uliana, Silvia R. B.
Date: 2021
Persistent ID: http://hdl.handle.net/10362/180764
Origin: Repositório Institucional da UNL
Subject(s): CRISPR/Cas9; endogenous tagging; knockout; Leishmania braziliensis; PF16; reverse genetics; Cell Biology; Genetics; Molecular Biology; Immunology; Microbiology; Parasitology; Infectious Diseases; Microbiology (medical); SDG 3 - Good Health and Well-being; SDG 9 - Industry, Innovation, and Infrastructure
Description
Funding Information: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2016/23405-4, 2018/25299-2, 2020/00087-2, 2018/14398-0, 2020/00088-9, 2016/00969-0, 2015/09080-2). SU is the recipient of a senior researcher scholarship from the Brazilian National Council for Scientific and Technological Development (CNPq 306971/2018-6). AC also acknowledges Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 and CNPq (305775/2013-8). AA-W acknowledges Fundação para a Ciência e a Tecnologia for funds to GHTM (UID/04413/2020). TB was supported by the Medical Research Council (15/16_MSD_836338), and EG is a Royal Society University Research Fellow and supported through the WCIP core Wellcome Centre Award No. 104111/Z/14/Z. Publisher Copyright: Copyright © 2021 Espada, Quilles, Albuquerque-Wendt, Cruz, Beneke, Lorenzon, Gluenz, Cruz and Uliana. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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.
