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

Author

Listed:
  • 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
    University of Texas Southwestern Medical Center)

  • Tae Hyun Hwang

    (Cleveland Clinic Lerner Research Institute)

  • Lawrence Lum

    (University of Texas Southwestern Medical Center)

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.

Suggested Citation

  • 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 & Lawrence Lum, 2019. "CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12028-5
    DOI: 10.1038/s41467-019-12028-5
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    Cited by:

    1. Chen Liu & Ioannis H. Hatzianestis & Thorsten Pfirrmann & Salim H. Reza & Elena A. Minina & Ali Moazzami & Simon Stael & Emilio Gutierrez–Beltran & Eugenia Pitsili & Peter Dörmann & Sabine D’Andrea & , 2024. "Seed longevity is controlled by metacaspases," Nature Communications, Nature, vol. 15(1), pages 1-17, December.

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