Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans

32 páginas, 4 figuras, 2 tablas. Más figuras y tablas como material suplementario

Detalles Bibliográficos
Autores: Vicencio, Jeremy, Martínez-Fernández, Carmen, Serrat, Xènia, Cerón, Julián
Tipo de recurso: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2019
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/414177
Acceso en línea:http://hdl.handle.net/10261/414177
Access Level:acceso abierto
Palabra clave:CRISPR
Caenorhabditis elegans
Cas9
Fluorescent proteins
Genome editing
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spelling Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegansVicencio, JeremyMartínez-Fernández, CarmenSerrat, XèniaCerón, JuliánCRISPRCaenorhabditis elegansCas9Fluorescent proteinsGenome editing32 páginas, 4 figuras, 2 tablas. Más figuras y tablas como material suplementarioCRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.This work has been supported by a grant from the Instituto de Salud Carlos III (ISCIII) to J.C. (PI15-00895), cofunded by FEDER funds/European Regional Development Fund (ERDF) — a way to Build Europe. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 713673. We thank CERCA (Centres de Recerca de Catalunya) Program/Generalitat de Catalunya for their institutional support. J.V. has an INPhINIT PhD fellowship from “la Caixa” Foundation (LCF/BQ/IN17/11620065), and X.S. has an FPU (Formación de Personal Universitario) Ph.D. fellowship from MINECO.Peer reviewedOxford University PressInstituto de Salud Carlos IIIEuropean CommissionEuropean Research CouncilCentres de Recerca de CatalunyaFundación la CaixaMinisterio de Economía y Competitividad (España)Cerón, Julián [0000-0003-4739-2243]202620262019info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/414177reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/H2020/713673https://doi.org/10.1534/genetics.119.301965Noinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4141772026-05-22T06:33:51Z
dc.title.none.fl_str_mv Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
title Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
spellingShingle Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
Vicencio, Jeremy
CRISPR
Caenorhabditis elegans
Cas9
Fluorescent proteins
Genome editing
title_short Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
title_full Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
title_fullStr Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
title_full_unstemmed Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
title_sort Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
dc.creator.none.fl_str_mv Vicencio, Jeremy
Martínez-Fernández, Carmen
Serrat, Xènia
Cerón, Julián
author Vicencio, Jeremy
author_facet Vicencio, Jeremy
Martínez-Fernández, Carmen
Serrat, Xènia
Cerón, Julián
author_role author
author2 Martínez-Fernández, Carmen
Serrat, Xènia
Cerón, Julián
author2_role author
author
author
dc.contributor.none.fl_str_mv Instituto de Salud Carlos III
European Commission
European Research Council
Centres de Recerca de Catalunya
Fundación la Caixa
Ministerio de Economía y Competitividad (España)
Cerón, Julián [0000-0003-4739-2243]
dc.subject.none.fl_str_mv CRISPR
Caenorhabditis elegans
Cas9
Fluorescent proteins
Genome editing
topic CRISPR
Caenorhabditis elegans
Cas9
Fluorescent proteins
Genome editing
description 32 páginas, 4 figuras, 2 tablas. Más figuras y tablas como material suplementario
publishDate 2019
dc.date.none.fl_str_mv 2019
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/414177
url http://hdl.handle.net/10261/414177
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/H2020/713673
https://doi.org/10.1534/genetics.119.301965
No
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Oxford University Press
publisher.none.fl_str_mv Oxford University Press
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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