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Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling

Benetó, Noelia ; Grinberg, Daniel ; Vilageliu, Lluïsa and Canals, Isaac LU (2022) In Methods in Molecular Biology 2549. p.409-425
Abstract

The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions. Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in... (More)

The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions. Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.

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Please use this url to cite or link to this publication:
author
; ; and
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
CRISPR/Cas9, Disease modeling, Genome editing, Human pluripotent stem cells
host publication
Methods in Molecular Biology
series title
Methods in Molecular Biology
volume
2549
pages
17 pages
publisher
Humana Press
external identifiers
  • pmid:33755903
  • scopus:85133547604
ISSN
1940-6029
1064-3745
DOI
10.1007/7651_2021_374
language
English
LU publication?
yes
id
b1ae57d9-c2cb-4e54-b063-7d354e9551d7
date added to LUP
2022-09-06 14:03:18
date last changed
2024-12-27 16:13:22
@inbook{b1ae57d9-c2cb-4e54-b063-7d354e9551d7,
  abstract     = {{<p>The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions. Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.</p>}},
  author       = {{Benetó, Noelia and Grinberg, Daniel and Vilageliu, Lluïsa and Canals, Isaac}},
  booktitle    = {{Methods in Molecular Biology}},
  issn         = {{1940-6029}},
  keywords     = {{CRISPR/Cas9; Disease modeling; Genome editing; Human pluripotent stem cells}},
  language     = {{eng}},
  pages        = {{409--425}},
  publisher    = {{Humana Press}},
  series       = {{Methods in Molecular Biology}},
  title        = {{Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling}},
  url          = {{http://dx.doi.org/10.1007/7651_2021_374}},
  doi          = {{10.1007/7651_2021_374}},
  volume       = {{2549}},
  year         = {{2022}},
}