RT Journal Article
SR Electronic
T1 CRISPR/Cas9-based mutagenesis frequently provokes on-target mRNA misregulation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 583138
DO 10.1101/583138
A1 Rubina Tuladhar
A1 Yunku Yeu
A1 John Tyler Piazza
A1 Zhen Tan
A1 Jean Rene Clemenceau
A1 Xiaofeng Wu
A1 Quinn Barrett
A1 Jeremiah Herbert
A1 David H. Mathews
A1 James Kim
A1 Tae Hyun Hwang
A1 Lawrence Lum
YR 2019
UL http://biorxiv.org/content/early/2019/03/20/583138.abstract
AB 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.