A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi

Autores
Vilchez Larrea, Salomé Catalina; Prego, Alejo Facundo; Schoijet, Alejandra Cecilia; Llanos, Manuel; Alberca, Lucas Nicolás; Bellera, Carolina Leticia; Gavernet, Luciana; Talevi, Alan; Alonso, Guillermo Daniel
Año de publicación
2022
Idioma
español castellano
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies. Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to the target gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a "pre-donor" formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This "pre-donor" allowed to easily generate a variety of donor DNAs by cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using "mixed" primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines.
Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Prego, Alejo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Llanos, Manuel. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina
Fil: Alberca, Lucas Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Bellera, Carolina Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Gavernet, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Talevi, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
XI Congreso de la Sociedad Argentina de Protozoología
Mendoza
Argentina
Sociedad Argentina de Protozoología
Materia
Chagas disease
Trypanosoma cruzi
CRISPR/Cas9
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/211753
Acceder
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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A versatile CRISPR/Cas9 editing approach in Trypanosoma cruziVilchez Larrea, Salomé CatalinaPrego, Alejo FacundoSchoijet, Alejandra CeciliaLlanos, ManuelAlberca, Lucas NicolásBellera, Carolina LeticiaGavernet, LucianaTalevi, AlanAlonso, Guillermo DanielChagas diseaseTrypanosoma cruziCRISPR/Cas9https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies. Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to the target gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a "pre-donor" formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This "pre-donor" allowed to easily generate a variety of donor DNAs by cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using "mixed" primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Prego, Alejo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Llanos, Manuel. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Alberca, Lucas Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Bellera, Carolina Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Gavernet, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Talevi, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXI Congreso de la Sociedad Argentina de ProtozoologíaMendozaArgentinaSociedad Argentina de ProtozoologíaUniversidad Nacional de Cuyo2022info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectCongresoJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/211753A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi; XI Congreso de la Sociedad Argentina de Protozoología; Mendoza; Argentina; 2022; 124-1241669-8991CONICET DigitalCONICETspainfo:eu-repo/semantics/altIdentifier/url/https://bdigital.uncu.edu.ar/app/navegador/?idobjeto=17095Internacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:04:28Zoai:ri.conicet.gov.ar:11336/211753instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:04:28.958CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
title A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
spellingShingle A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
Vilchez Larrea, Salomé Catalina
Chagas disease
Trypanosoma cruzi
CRISPR/Cas9
title_short A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
title_full A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
title_fullStr A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
title_full_unstemmed A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
title_sort A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi
dc.creator.none.fl_str_mv Vilchez Larrea, Salomé Catalina
Prego, Alejo Facundo
Schoijet, Alejandra Cecilia
Llanos, Manuel
Alberca, Lucas Nicolás
Bellera, Carolina Leticia
Gavernet, Luciana
Talevi, Alan
Alonso, Guillermo Daniel
author Vilchez Larrea, Salomé Catalina
author_facet Vilchez Larrea, Salomé Catalina
Prego, Alejo Facundo
Schoijet, Alejandra Cecilia
Llanos, Manuel
Alberca, Lucas Nicolás
Bellera, Carolina Leticia
Gavernet, Luciana
Talevi, Alan
Alonso, Guillermo Daniel
author_role author
author2 Prego, Alejo Facundo
Schoijet, Alejandra Cecilia
Llanos, Manuel
Alberca, Lucas Nicolás
Bellera, Carolina Leticia
Gavernet, Luciana
Talevi, Alan
Alonso, Guillermo Daniel
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Chagas disease
Trypanosoma cruzi
CRISPR/Cas9
topic Chagas disease
Trypanosoma cruzi
CRISPR/Cas9
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies. Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to the target gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a "pre-donor" formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This "pre-donor" allowed to easily generate a variety of donor DNAs by cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using "mixed" primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines.
Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Prego, Alejo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Llanos, Manuel. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina
Fil: Alberca, Lucas Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Bellera, Carolina Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Gavernet, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Talevi, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina
Fil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
XI Congreso de la Sociedad Argentina de Protozoología
Mendoza
Argentina
Sociedad Argentina de Protozoología
description Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies. Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to the target gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a "pre-donor" formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This "pre-donor" allowed to easily generate a variety of donor DNAs by cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using "mixed" primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines.
publishDate 2022
dc.date.none.fl_str_mv 2022
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http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/211753
A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi; XI Congreso de la Sociedad Argentina de Protozoología; Mendoza; Argentina; 2022; 124-124
1669-8991
CONICET Digital
CONICET
url http://hdl.handle.net/11336/211753
identifier_str_mv A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi; XI Congreso de la Sociedad Argentina de Protozoología; Mendoza; Argentina; 2022; 124-124
1669-8991
CONICET Digital
CONICET
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publisher.none.fl_str_mv Universidad Nacional de Cuyo
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