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Methods. 2017 May 15;121-122:45-54. doi: 10.1016/j.ymeth.2017.05.003. Epub 2017 May 10.

Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

Author information

1
Institut Curie, PSL Research University, 75005 Paris, France; INSERM U934, Paris, France; CNRS UMR3215, Paris, France. Electronic address: michel.wassef@curie.fr.
2
INSERM UMR_S745 et EA7331, Université Paris Descartes, Sorbonne Paris Cité, Facultée des Sciences Pharmaceutiques et Biologiques, 75006 Paris, France; Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, 75014 Paris, France.
3
Institut Curie, PSL Research University, 75005 Paris, France; INSERM U934, Paris, France; CNRS UMR3215, Paris, France.
4
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.
5
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA; University of Florida Health Cancer Center, University of Florida, Gainesville, FL, USA; University of Florida Genetics Institute, University of Florida, Gainesville, FL, USA.

Abstract

The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.

KEYWORDS:

CRISPR/CAS9; Constitutive knockout; Genome editing; Point mutation; Reversible knockout; Targeting vector

PMID:
28499832
DOI:
10.1016/j.ymeth.2017.05.003
[Indexed for MEDLINE]

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