Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing

CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, bas...

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Autores: Aguilar, Gustavo, Bauer, Milena, Vigano, M Alessandra, Schnider, Sophie T, Brügger, Lukas, Jiménez-Jiménez, Carlos, Guerrero, Isabel, Affolter, Markus
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2024
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/387853
Acceso en línea:http://hdl.handle.net/10261/387853
Access Level:acceso abierto
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spelling Seamless knockins in Drosophila via CRISPR-triggered single-strand annealingAguilar, GustavoBauer, MilenaVigano, M AlessandraSchnider, Sophie TBrügger, LukasJiménez-Jiménez, CarlosGuerrero, IsabelAffolter, MarkusCRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from “scarless editing by element deletion”), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.The work in the laboratory of M.A. was supported by grants from the Swiss National Science Foundation (310030_192659/1) and by funds from the Kanton Basel-Stadt and Basel-Land. G.A., M.B., and S.S. were supported by ‘‘Fellowships for Excellence’’ from the International PhD Program in Molecular Life Sciences of the Biozentrum, University of Basel. The work at the I.G. laboratory was supported by grants PID2020-114533GB-C21 to I.G. and PRE2018-085510 to C.J.-J. from the Spanish Ministry of Science, Innovation and Universities.Swiss National Science FoundationMinisterio de Ciencia, Innovación y Universidades (España)Agencia Estatal de Investigación (España)University of BaselConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2025202520242025info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/387853reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-114533GB-C21http://dx.doi.org/10.1016/j.devcel.2024.06.004Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3878532026-05-22T06:33:51Z
dc.title.none.fl_str_mv Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
title Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
spellingShingle Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
Aguilar, Gustavo
title_short Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
title_full Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
title_fullStr Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
title_full_unstemmed Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
title_sort Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing
dc.creator.none.fl_str_mv Aguilar, Gustavo
Bauer, Milena
Vigano, M Alessandra
Schnider, Sophie T
Brügger, Lukas
Jiménez-Jiménez, Carlos
Guerrero, Isabel
Affolter, Markus
author Aguilar, Gustavo
author_facet Aguilar, Gustavo
Bauer, Milena
Vigano, M Alessandra
Schnider, Sophie T
Brügger, Lukas
Jiménez-Jiménez, Carlos
Guerrero, Isabel
Affolter, Markus
author_role author
author2 Bauer, Milena
Vigano, M Alessandra
Schnider, Sophie T
Brügger, Lukas
Jiménez-Jiménez, Carlos
Guerrero, Isabel
Affolter, Markus
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Swiss National Science Foundation
Ministerio de Ciencia, Innovación y Universidades (España)
Agencia Estatal de Investigación (España)
University of Basel
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
description CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from “scarless editing by element deletion”), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
publishDate 2024
dc.date.none.fl_str_mv 2024
2025
2025
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/387853
url http://hdl.handle.net/10261/387853
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/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-114533GB-C21
http://dx.doi.org/10.1016/j.devcel.2024.06.004
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
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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