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

May 15, 2019

Received Date 6th May 19

The introduction of insertion-deletions (INDELs) by activation of the error-prone non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR/Cas9-directed genome editing. The ability of CRISPR/Cas9 to achieve gene elimination (knockouts) is largely attributed to the emergence of a pre-mature termination codon (PTC) from a frameshift-inducing INDEL that elicits non-sense mediated decay (NMD) of the mutant mRNA. Yet, the impact on gene expression as a consequence of CRISPR/Cas9-introduced INDELs into RNA regulatory sequences has been largely left uninvestigated. By tracking DNA-mRNA-protein relationships in a collection of CRISPR/Cas9-edited cell lines that harbor frameshift-inducing INDELs in various targeted genes, we detected the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products are derived from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results using CRISPR/Cas9-introduced INDELs reveal facets of an epigenetic genome buffering apparatus that likely evolved to mitigate the impact of such mutations introduced by pathogens and aberrant DNA damage repair, and that more recently pose challenges to manipulating gene expression outcomes using INDEL-based mutagenesis.

Read in full at bioRxiv.

This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.

Nature Communications

Nature Research, Springer Nature