Increased mRNA expression after CRISPR-Cas9 (intended loss-of-function) editing?

Dear Colleague,

I hope this message finds you well. Observing increased mRNA expression following CRISPR-Cas9 editing intended to create a loss-of-function mutation can be perplexing. Several factors may contribute to this unexpected result. Here, I provide a detailed and logical analysis to help you understand and address this issue:

Incomplete Knockout:Partial Editing: The CRISPR-Cas9 system might not have efficiently knocked out both alleles of the target gene, leading to residual expression. Verify the editing efficiency by sequencing the target site in multiple clones to ensure both alleles have been disrupted. Alternative Splicing: CRISPR-induced indels can lead to alternative splicing, potentially creating transcripts that are stable but non-functional. Examine the mRNA sequence for alternative splicing events that might result from the editing.

Compensatory Mechanisms:Feedback Regulation: Loss of function in one gene can trigger compensatory upregulation of other genes or the target gene itself due to feedback mechanisms. Investigate whether compensatory pathways are activated by analyzing the expression of related genes or pathways. Transcriptional Activation: In some cases, CRISPR-Cas9 editing might inadvertently disrupt repressive regulatory elements, leading to increased transcription of the target gene. Assess the genomic region for potential regulatory elements that might have been affected by the editing.

Non-Specific Effects:Off-Target Effects: CRISPR-Cas9 can induce off-target mutations that might affect the expression of the target gene or other genes involved in its regulation. Perform whole-genome sequencing or targeted off-target analysis to identify any off-target effects. Transcriptional Noise: Editing can sometimes lead to transient increases in transcriptional noise, resulting in higher variability and apparent upregulation of the target gene. Repeat the experiment and analyze multiple biological replicates to confirm the findings.

Technical Artifacts:qRT-PCR Primer Design: Ensure that the qRT-PCR primers used for measuring mRNA expression are specific to the intended target and do not amplify related sequences or pseudogenes. RNA Integrity: Verify the integrity and quality of the RNA samples to rule out degradation or contamination that might affect the qRT-PCR results. Use internal controls and housekeeping genes to normalize the data accurately.

Cellular Context:Cell Type-Specific Responses: Different cell types may respond differently to CRISPR-Cas9 editing due to their unique transcriptional and regulatory landscapes. Consider repeating the experiment in multiple cell lines to verify the results. Stress Responses: CRISPR-Cas9 editing can induce cellular stress responses that might transiently increase mRNA expression. Monitor stress-related markers and minimize stress during the experimental procedures.

To address the observed increase in mRNA expression, I recommend the following steps:

Validation: Confirm the editing efficiency and the nature of the indels by sequencing the target locus.
Replication: Perform the experiment with multiple independent clones and biological replicates to ensure consistency.
Comprehensive Analysis: Analyze the expression of related genes and pathways to identify potential compensatory mechanisms.
Off-Target Assessment: Conduct off-target analysis to identify and mitigate any unintended effects.

By systematically addressing these factors, you can better understand the underlying cause of the increased mRNA expression and take appropriate measures to achieve the intended loss-of-function outcome.

Should you have any further questions or require additional assistance, please feel free to reach out.

This list of protocols might help us better address the issue.

Stefan Anchev Kirov

What are you targeting precisely? Is there a reason to expect decrease in mRNA beyond as a result of non-sense mediated decay?

Do you see the editing event in the mRNA (if you did RNASeq)?

Here is a paper you may want to look at as well:

Article Unexpected consequences: Exon skipping caused by CRISPR-gene...

Jane Lindborg

Thanks for the answers. I am in the process of checking protein expression. But it still doesn't quite address why the edited neurons produce detectable transcript levels for a few of the targets while the control samples are undetectable. I might just start new cultures and try the assay again.

Jane,

Replicating the results is always a good idea.

If you see again detectable levels, perhaps you should sequence the transcript to make sure the ORF is disrupted.

Just out of curiosity- why are you knocking down genes that are not expressed?

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