Epigenetic Mechanism May Limit CRISPR Effectiveness

CRISPR technology has been one of the hottest tools to emerge in genetics research. Developed based on the modes of defense used by bacteria, CRISPR/Cas9 proteins destroy pathogen DNA in order to prevent the bacterial cells. Scientists were able to then leverage this ability into a tool that can be used to edit genes so that DNA sequences could be altered directly to modify gene expression.

The introduction of CRISPR/Cas9 into the biological research toolbox has been revolutionary, primarily in terms of the timescale of these edits; instead of needing to wait weeks or months for edits to be successfully made, CRISPR can make it happen in a matter of days.

But the use of CRISPR technology has not come without obstacles. DNA is tightly packaged in a complex called chromatin, making it difficult for transcriptional access. It can be vulnerable to histone methylation, an epigenetic mechanism that can affect gene expression.

Histone methylation involves the addition of a methyl group to certain amino acids of a histone protein. This typically results in the further tightening of DNA, which could inhibit CRISPR function and obstruct the desired edits from being made. Researchers need a way to specifically loosen chromatin during CRISPR applications in order to successfully perform the desired edits.

A joint bioengineering collaboration between Arizona State University and Emory University looked at different mechanisms that could help improve the efficiency of CRISPR/Cas9 machinery—focusing on different molecular and epigenetic inhibitors.

The team developed protein editorial assistants that can undo this chromatin blocking. When delivered alongside the CRISPR editing machinery, these protein assistants can help remove the obstructions created by the chromatin—making the DNA sites accessible, and allowing for successful editing.

“The idea is that if CRISPR needs to bind in the middle of a gene but can’t bind close enough to edit the mutation, you could send in our chromatin-opening protein to right outside that hard-to-bind region, rearrange the chromatin, and make the DNA across that gene more accessible for CRISPR to edit the gene,” says lead author Karmella Haynes.

The research team headed by Dr. Haynes determined that H3K27me3 played a role in the tightening of chromatin, and sought to reverse its effect. They used a system where altering chromatin accessibility could switch a gene called luciferase on or off, which would turn associated luminescence on or off and making it easy to check whether luciferase was silenced or not.

After testing several activation-associated proteins (AAPs), they were able to find specific ones that could alter the chromatin and leave the luciferase gene exposed to the CRISPR editing machinery.

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The extent to which the silencing induced by chromatin blocking was undone differed with the AAPs used; only Gal4P65 totally restored the luciferase function, making it the ideal protein assistant for this particular application. They found that although H3K27me3 could be successfully removed, it could not successfully reactivate transcription.  

This research therefore also suggests that protein assistants might need to be cataloged and used specifically for each particular intended CRISPR application. Further research is needed to explore how these protein assistants should work, but it is promising that CRISPR’s efficacy can be heightened.

Dr. Haynes went on to express hopes for the future next steps of this research: “It’d be interesting to find out whether one type of AAP is more effective at disrupting chromatin at some genes versus others. Or whether combining proteins together might further enhance CRISPR editing. I envision there being a whole catalog of CRISPR cofactors that can be used to enhance CRISPR activity.”

Source: Haynes K, et al. (2020) Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity APL Bioengineering 4

Reference: APL Bioengineering “Opening Up DNA to Delete Disease” APL Bioengineering News. 14 Jan 2020. Web.

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About Andrea P 30 Articles
Andrea received her B.S. in Biology with minors in Chemistry and Neuroscience from Duke University. She first fell in love with biology when she learned about the magnificent powers of protein folding, and then naturally wanted to know who was in charge. She’s fascinated by the finer controls of epigenetic modifications. In her downtime, she enjoys hiking with her dog and going for long drives to explore new places.

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