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Nat Commun. 2019 Sep 6;10(1):4056. doi: 10.1038/s41467-019-12028-5.

CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation.

Author information

1
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
2
Department of Quantitative Health Sciences, Cleveland Clinic Lerner Research Institute, Cleveland, OH, 44195, USA.
3
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, 14642, USA.
4
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
5
Hamon Center for Therapeutic Oncology Research Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
6
Department of Quantitative Health Sciences, Cleveland Clinic Lerner Research Institute, Cleveland, OH, 44195, USA. hwangt@ccf.org.
7
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. lawrence.lum@utsouthwestern.edu.

Abstract

The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.

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