Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Autor(es)

Lander, Noelia ^[1] ; Chiurillo, Miguel A. ^[1] ; Vercesi, Anibal E. ^[1] ; Docampo, Roberto ^{[2, 3, 1]}

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Fac Ciencias Med, Dept Patol Clin, BR-13083 Campinas, SP - Brazil

^[2] Univ Georgia, Ctr Trop & Emerging Global Dis, Athens, GA 30602 - USA

^[3] Univ Georgia, Dept Cellular Biol, Athens, GA 30602 - USA

• Tipo de documento: Artigo Científico
• Fonte: BIO-PROTOCOL; v. 7, n. 10 MAY 20 2017.
• Citações Web of Science: 7

Resumo

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis. (AU)

• Processo FAPESP: 14/08995-4 - Sinalização por íons de cálcio em tripanosomatídeos
• Beneficiário: Noelia Marina Lander Manfredi
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 14/13148-9 - Sinalização por íons de cálcio em tripanossomatídeos
• Beneficiário: Miguel Angel Chiurillo Siervo
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 13/50624-0 - Sinalização por íons de cálcio em tripanossomatídeos
• Beneficiário: Roberto Docampo
• Modalidade de apoio: Auxílio à Pesquisa - Programa SPEC