Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

[en] Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2 % of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15 % efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

Grobarczyk, Benjamin ; Université de Liège > GIGA - Neurosciences

Franco, Bénédicte ; Université de Liège > GIGA - Neurosciences

Hanon, Kevin

Malgrange, Brigitte ; Université de Liège > GIGA - Neurosciences

Language :

English

Title :

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

Publication date :

2015

Journal title :

Stem Cell Reviews

ISSN :

1550-8943

eISSN :

1558-6804

Publisher :

Springer, Germany

Peer reviewed :

Peer Reviewed verified by ORBi

Funders :

F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
Fonds Léon Fredericq [BE]
BELSPO - SPP Politique scientifique - Service Public Fédéral de Programmation Politique scientifique

Available on ORBi :

since 12 June 2015

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