Navegando por Palavras-chave "CRISPR-Cas9"

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    Raising the Bar(-seq) in Leishmania genetic screens
    (Cell Press, 2021) Ferreira, Tiago R.; Couñago, Rafael M.; Moretti, Nilmar Silvio [UNIFESP]; http://lattes.cnpq.br/2131472726202687
    Our understanding of regulatory factors in Leishmania differentia- tion has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.
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    Use of LeishGEdit CRISPR-Cas9 technology for genetic manipulation of protozoan parasite Leishmania
    (JoVE, 2021) Maran, Suellen Rodrigues [UNIFESP]; Bonifácio, Bruno Souza [UNIFESP]; Zanchetta, Myrna [UNIFESP]; Garcia, Miguel Antonio do Nascimento [UNIFESP]; Catta-Preta, Carolina M. C.; Moretti, Nilmar Silvio [UNIFESP]; http://lattes.cnpq.br/2131472726202687
    The cell biology of a parasitic protozoan as well as the impact of the infection in host cells can be addressed using genome modification techniques. The development of robust methods eases the burden to obtain gene mutants and contributes to answer specific biological questions. Here we describe the LeishGEdit CRISPR-Cas9 high- throughput method that allows for Leishmania in situ gene tagging and deletion in a short span of time (7-10 days). Briefly, a transgenic cell line expressing SpCas9 and T7 RNA polymerase serves as the background for electroporation of DNA fragments generated by PCR: (1) a fragment containing a T7 promoter and the gene specific guide RNA expressed with a Cas9 scaffold; and (2) a homologous recombination (HR) fragment to introduce a resistance marker and/or a fluorescent tag/epitope to the desired genome location. Our protocol will cover (1) primer design, (2) DNA fragment production and confirmation, (3) transfection, and (4) cell line confirmation methods. We hope the article will allow for easy reproduction of the protocol for genome manipulation by CRISPR-Cas9 and make the method largely available to the parasitology community, enabling advances in the understanding of the biology of Leishmania and other protozoan pathogens of medical and veterinary importance.