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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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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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. |
format | Online Article Text |
id | pubmed-6481050 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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