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CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Reber Stefan, Mechtersheimer Jonas, Nasif Sofia, Benitez Julio Aguila, Colombo Martino, Domanski Michal, Jutzi Daniel, Hedlund Eva, Ruepp Marc-David,
Project Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD)
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Original article (peer-reviewed)

Journal Molecular Biology of the Cell
Volume (Issue) 29(2)
Page(s) 75 - 83
Title of proceedings Molecular Biology of the Cell
DOI 10.1091/mbc.e17-05-0288

Open Access


CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to ∼50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.