Cargando…

Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors

Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genome...

Descripción completa

Detalles Bibliográficos
Autores principales: Paix, Alexandre, Rasoloson, Dominique, Folkmann, Andrew, Seydoux, Geraldine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935516/
https://www.ncbi.nlm.nih.gov/pubmed/31710422
http://dx.doi.org/10.1002/cpmb.102
_version_ 1783483589048926208
author Paix, Alexandre
Rasoloson, Dominique
Folkmann, Andrew
Seydoux, Geraldine
author_facet Paix, Alexandre
Rasoloson, Dominique
Folkmann, Andrew
Seydoux, Geraldine
author_sort Paix, Alexandre
collection PubMed
description Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology‐directed repair of DNA breaks induced by the Cas9 RNA‐guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30‐40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9‐crRNA‐tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.
format Online
Article
Text
id pubmed-6935516
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher John Wiley and Sons Inc.
record_format MEDLINE/PubMed
spelling pubmed-69355162020-12-01 Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors Paix, Alexandre Rasoloson, Dominique Folkmann, Andrew Seydoux, Geraldine Curr Protoc Mol Biol Updated Protocol Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology‐directed repair of DNA breaks induced by the Cas9 RNA‐guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30‐40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9‐crRNA‐tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors. John Wiley and Sons Inc. 2019-09-19 2019-12 /pmc/articles/PMC6935516/ /pubmed/31710422 http://dx.doi.org/10.1002/cpmb.102 Text en © 2019 The Authors. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Updated Protocol
Paix, Alexandre
Rasoloson, Dominique
Folkmann, Andrew
Seydoux, Geraldine
Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title_full Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title_fullStr Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title_full_unstemmed Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title_short Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors
title_sort rapid tagging of human proteins with fluorescent reporters by genome engineering using double‐stranded dna donors
topic Updated Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935516/
https://www.ncbi.nlm.nih.gov/pubmed/31710422
http://dx.doi.org/10.1002/cpmb.102
work_keys_str_mv AT paixalexandre rapidtaggingofhumanproteinswithfluorescentreportersbygenomeengineeringusingdoublestrandeddnadonors
AT rasolosondominique rapidtaggingofhumanproteinswithfluorescentreportersbygenomeengineeringusingdoublestrandeddnadonors
AT folkmannandrew rapidtaggingofhumanproteinswithfluorescentreportersbygenomeengineeringusingdoublestrandeddnadonors
AT seydouxgeraldine rapidtaggingofhumanproteinswithfluorescentreportersbygenomeengineeringusingdoublestrandeddnadonors