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

Methods : a Companion to Methods in Enzymology
M WassefR Margueron

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,...Continue Reading

Citations

Jan 22, 2019·Nature Communications·Antoine CampagneRaphaël Margueron
Mar 4, 2020·Expert Review of Molecular Diagnostics·Bruna Dalmasso, Paola Ghiorzo
May 10, 2019·PloS One·Ignacio García-TuñónManuel Sánchez-Martín
May 19, 2020·EMBO Reports·Patricia M DavidsonCécile Sykes
Dec 14, 2019·Molecular Biology Reports·Ignacio García-TuñónManuel Sánchez-Martín
Aug 25, 2018·Investigative Ophthalmology & Visual Science·Kumari Alka, Joseph R Casey
Dec 10, 2020·Life Science Alliance·Arnaud RondeletAlexander W Bird
Jan 5, 2021·DNA and Cell Biology·Jieyu LiuGui-Lan Chen
Feb 10, 2021·Biology·Elena VueltaManuel Sánchez-Martín
May 6, 2019·Methods : a Companion to Methods in Enzymology·Aleksandra SpiegelMihail Sarov

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