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Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2...

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Autores principales: Moir-Meyer, Gemma, Cheong, Pak Leng, Olijnik, Aude-Anais, Brown, Jill, Knight, Samantha, King, Andrew, Kurita, Ryo, Nakamura, Yukio, Gibbons, Richard J., Higgs, Douglas R., Buckle, Veronica J., Babbs, Christian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481050/
https://www.ncbi.nlm.nih.gov/pubmed/31164570
http://dx.doi.org/10.3390/mps1030028
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author Moir-Meyer, Gemma
Cheong, Pak Leng
Olijnik, Aude-Anais
Brown, Jill
Knight, Samantha
King, Andrew
Kurita, Ryo
Nakamura, Yukio
Gibbons, Richard J.
Higgs, Douglas R.
Buckle, Veronica J.
Babbs, Christian
author_facet Moir-Meyer, Gemma
Cheong, Pak Leng
Olijnik, Aude-Anais
Brown, Jill
Knight, Samantha
King, Andrew
Kurita, Ryo
Nakamura, Yukio
Gibbons, Richard J.
Higgs, Douglas R.
Buckle, Veronica J.
Babbs, Christian
author_sort Moir-Meyer, Gemma
collection PubMed
description The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.
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spelling pubmed-64810502019-05-31 Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors Moir-Meyer, Gemma Cheong, Pak Leng Olijnik, Aude-Anais Brown, Jill Knight, Samantha King, Andrew Kurita, Ryo Nakamura, Yukio Gibbons, Richard J. Higgs, Douglas R. Buckle, Veronica J. Babbs, Christian Methods Protoc Protocol The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations. MDPI 2018-07-30 /pmc/articles/PMC6481050/ /pubmed/31164570 http://dx.doi.org/10.3390/mps1030028 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Moir-Meyer, Gemma
Cheong, Pak Leng
Olijnik, Aude-Anais
Brown, Jill
Knight, Samantha
King, Andrew
Kurita, Ryo
Nakamura, Yukio
Gibbons, Richard J.
Higgs, Douglas R.
Buckle, Veronica J.
Babbs, Christian
Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title_full Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title_fullStr Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title_full_unstemmed Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title_short Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
title_sort robust crispr/cas9 genome editing of the hudep-2 erythroid precursor line using plasmids and single-stranded oligonucleotide donors
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481050/
https://www.ncbi.nlm.nih.gov/pubmed/31164570
http://dx.doi.org/10.3390/mps1030028
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