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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • CRISPR-Cas Systems
  • Cell Line
  • Cell Line, Tumor
  • Codon, Nonsense / genetics
  • Frameshift Mutation
  • Gene Editing / methods*
  • Gene Expression Regulation, Neoplastic
  • Gene Knockout Techniques
  • HeLa Cells
  • Humans
  • INDEL Mutation
  • Mutagenesis*
  • RNA Stability
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics*

Substances

  • Codon, Nonsense
  • RNA, Messenger