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dc.contributor.authorMaran, Suellen Rodrigues [UNIFESP]
dc.contributor.authorBonifácio, Bruno Souza [UNIFESP]
dc.contributor.authorZanchetta, Myrna [UNIFESP]
dc.contributor.authorGarcia, Miguel Antonio do Nascimento [UNIFESP]
dc.contributor.authorCatta-Preta, Carolina M. C.
dc.contributor.authorMoretti, Nilmar Silvio [UNIFESP]
dc.date.accessioned2021-11-29T16:13:55Z
dc.date.available2021-11-29T16:13:55Z
dc.date.issued2021
dc.identifier.urihttps://repositorio.unifesp.br/xmlui/handle/11600/62327
dc.description.abstractThe 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.pt_BR
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.languageengpt_BR
dc.publisherJoVEpt_BR
dc.relation.ispartofJoVE Journal of Visualized Experimentspt_BR
dc.rightsAcesso restritopt_BR
dc.subjectCRISPR-Cas9pt_BR
dc.subjectLeishmaniasispt_BR
dc.subjectLeishmaniapt_BR
dc.titleUse of LeishGEdit CRISPR-Cas9 technology for genetic manipulation of protozoan parasite Leishmaniapt_BR
dc.typeArtigopt_BR
dc.description.sponsorshipIDFAPESP: 2018/09948-0pt_BR
dc.description.sponsorshipIDFAPESP: 2019/13765-1pt_BR
dc.description.sponsorshipIDFAPESP: 2020/01434-8pt_BR
dc.description.sponsorshipIDCNPq: 424729/2018pt_BR
dc.identifier.doi10.3791/62297
unifesp.campusEscola Paulista de Medicina (EPM)pt_BR
unifesp.graduateProgramMicrobiologia e Imunologiapt_BR
unifesp.knowledgeAreaOutrapt_BR
dc.contributor.authorLatteshttp://lattes.cnpq.br/2131472726202687pt_BR
unifesp.departamentoMicrobiologia, Imunologia e Parasitologiapt_BR


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