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

Rubina Tuladhar, Yunku Yeu, John Tyler Piazza, Zhen Tan, Jean Rene Clemenceau, Xiaofeng Wu, Quinn Barrett, Jeremiah Herbert, David H. Mathews, James Kim, Tae Hyun Hwang () and Lawrence Lum ()
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Rubina Tuladhar: University of Texas Southwestern Medical Center
Yunku Yeu: Cleveland Clinic Lerner Research Institute
John Tyler Piazza: University of Texas Southwestern Medical Center
Zhen Tan: University of Rochester Medical Center
Jean Rene Clemenceau: Cleveland Clinic Lerner Research Institute
Xiaofeng Wu: University of Texas Southwestern Medical Center
Quinn Barrett: University of Texas Southwestern Medical Center
Jeremiah Herbert: University of Texas Southwestern Medical Center
David H. Mathews: University of Rochester Medical Center
James Kim: University of Texas Southwestern Medical Center
Tae Hyun Hwang: Cleveland Clinic Lerner Research Institute
Lawrence Lum: University of Texas Southwestern Medical Center

Nature Communications, 2019, vol. 10, issue 1, 1-10

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-12028-5

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