CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation
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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
Rubina Tuladhar1, Yunku Yeu2, John Tyler Piazza1
1Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
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.
Insights
CRISPR-Cas9 genome editing using insertion-deletions (INDELs) can create unintended foreign mRNAs and proteins in about 50% of cell lines. This challenges precise gene knockout strategies and requires new mitigation approaches.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing relies on non-homologous end-joining (NHEJ) to introduce insertion-deletions (INDELs).
- Gene ablation is typically achieved by INDELs causing frameshifts and premature termination codons (PTCs), leading to nonsense-mediated decay (NMD) of mRNA.
- However, the fidelity of CRISPR-Cas9-induced gene knockouts requires further investigation.
Purpose of the Study:
- To investigate the unintended consequences of CRISPR-Cas9-mediated INDEL mutagenesis on mRNA and protein production.
- To identify the mechanisms by which aberrant transcripts and proteins are generated despite presumed gene knockouts.
- To reveal challenges and propose strategies for improving the accuracy of CRISPR-Cas9 genome editing outcomes.
Main Methods:
- Analysis of mRNA and protein products from CRISPR-Cas9 targeted genes in a panel of cell lines.
- Examination of cell lines with presumed gene knockouts generated via CRISPR-Cas9.
- Investigation of INDEL-induced alterations in mRNA processing, including internal ribosomal entry, pseudo-mRNA conversion, and exon skipping.
Main Results:
- Approximately 50% of cell lines with presumed CRISPR-Cas9-induced gene knockouts produced foreign mRNAs or proteins.
- Aberrant protein products resulted from INDELs that facilitated internal ribosomal entry.
- INDELs also converted pseudo-mRNAs into protein-encoding molecules and induced exon skipping by disrupting exon splicing enhancers (ESEs).
Conclusions:
- CRISPR-Cas9-mediated INDEL mutagenesis presents significant challenges in achieving selective gene ablation due to the production of unintended mRNA and protein products.
- Mechanisms such as internal ribosomal entry, pseudo-mRNA conversion, and disrupted splicing contribute to these off-target effects.
- Understanding these challenges is crucial for developing strategies to mitigate their impact and enhance the precision of genome editing outcomes.
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