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, Lawrence Lum

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number4056
JournalNature communications
Volume10
Issue number1
DOIs
StatePublished - Dec 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
mutagenesis
deletion
Mutagenesis
insertion
Genes
genes
proteins
editing
Messenger RNA
genome
Nonsense Codon
cultured cells
Exons
Proteins
Nonsense Mediated mRNA Decay
Cell Line
splicing
Gene Knockout Techniques
Cells

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

Cite this

CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. / Tuladhar, Rubina; Yeu, Yunku; Tyler Piazza, John; Tan, Zhen; Rene Clemenceau, Jean; Wu, Xiaofeng; Barrett, Quinn; Herbert, Jeremiah; Mathews, David H.; Kim, James; Hyun Hwang, Tae; Lum, Lawrence.

In: Nature communications, Vol. 10, No. 1, 4056, 01.12.2019.

Research output: Contribution to journalArticle

Tuladhar, R, Yeu, Y, Tyler Piazza, J, Tan, Z, Rene Clemenceau, J, Wu, X, Barrett, Q, Herbert, J, Mathews, DH, Kim, J, Hyun Hwang, T & Lum, L 2019, 'CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation', Nature communications, vol. 10, no. 1, 4056. https://doi.org/10.1038/s41467-019-12028-5
Tuladhar, Rubina ; Yeu, Yunku ; Tyler Piazza, John ; Tan, Zhen ; Rene Clemenceau, Jean ; Wu, Xiaofeng ; Barrett, Quinn ; Herbert, Jeremiah ; Mathews, David H. ; Kim, James ; Hyun Hwang, Tae ; Lum, Lawrence. / CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. In: Nature communications. 2019 ; Vol. 10, No. 1.
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