Yale study details method to increase accuracy and safety of CRISPR gene editing

Yale’s Glazer Lab has published a study on the possibility of increasing the precision and safety of CRISPR-Cas9 gene editing through the use of peptide nucleic acids.

Elizabeth Watson

11:02 p.m., March 09, 2022

Staff reporter

Ellie Huh

A new study published at Yale’s Glazer Lab has important implications for improving the accuracy and efficiency of CRISPR-Cas9 gene editing.

The research team was led by Nicholas Economos MED ’23, a Glazer Lab MD-PhD candidate who was the paper’s first author, and Peter Glazer, a professor of therapeutic radiology. The study aimed to investigate a new way to increase the accuracy and safety of CRISPR-Cas9 gene editing through the use of peptide nucleic acids, called PNAs, to treat patients with genetic diseases.

“There was no precedent for this, and we had never done anything like this,” Economos said. “It was a really cool idea. Initially, when we were using [PNAs] as inhibitors and I first picked up that data, I was like ‘Wow, that’s a real effect. It always works and depends on the sequence, and I do all the checks and they all work. It was really a kind of moment of discovery, as I feel in the lab and especially in the doctorate. kind of situation is a rare thing to get.

CRISPR-Cas9 performs gene editing using the Cas9 enzyme, which cuts DNA at specific sequences identified by guide RNA and its homologous sequences. Because PNAs can bind strongly to DNA and RNA, Economos and his colleagues became curious about how PNAs might impact CRISPR if they were allowed to bind to the target sequence of 20 nucleotides called the guide RNA “spacer”, which helps guide the Cas9 enzyme. These are called anti-spacer PNAs, where “anti” refers to the binding that occurs between PNAs and RNA spacers. This led to a series of experiments in which Economos engineered various types of PNAs and combined them with different CRISPR target genes.

The experiments demonstrated that it is possible for PNAs to inhibit CRISPR-Cas9 activity by binding to a particular region of the guide RNA spacer, acting as a “switch” for the enzyme. This could play an important role in regulating the technology to prevent harm to patients. For example, to prevent off-target effects that could cause toxic events, ANP molecules could be delivered to specific organs to shut down Cas9 activity, according to Economos.

“As we begin to move towards getting CRISPR into the bloodstream and systemically treating people with CRISPR, there are issues with making sure the right organ system is targeted in this stuff.” , Economos said. “There is a very great need for a tool to monitor and improve the accuracy and safety of the use of this technology in humans, because at the end of the day there are certain dangers associated with the use of powerful technology. .”

The team also found that designing PNAs that tend to bind to particular regions of the guide RNA spacer could increase the precision of gene editing with CRISPR-Cas9. Different regions of the spacer can have different functions, so by designing PNAs with specific binding tendencies, it is possible to improve the precision of gene editing by ensuring that the desired effects take place.

It also increases the likelihood that CRISPR-Cas9 will reach and interact with its intended target site, while simultaneously reducing the possibilities of adverse off-target effects. An example given by Economos of a specific “off-target” effect that could occur would be Cas9 being mistakenly activated in the liver when other organ systems are intended targets. This could be solved by using PNA.

“[One application of this technology] is to modulate the activity of CRISPR-Cas9-like technologies,” Glazer said. “CRISPR can be used to cut DNA and mediate gene editing, but you can also modify the Cas9 protein and make it a fusion protein with factors that regulate gene expression. You can have what they call CRISPR activation or CRISPR inhibition.

This project was selected as a finalist for the 2022 Nucleate Activator Program, a national incubator that strives to develop the next generation of biotechnology and clinical treatment companies. Economos hopes to eventually apply the results of this venture to produce safer methods of gene editing for patients.

“This particular project is simply discovering something new and developing it so other people can use it and advance science as a whole and work to help people together in collaborative ways,” Kelly Carufe GRD ’24, student graduate at Glazer Lab, mentioned.

The Glazer Lab is part of the Yale Cancer Center’s Department of Therapeutic Radiology.


Elizabeth Watson covers groundbreaking research for SciTech and illustrates for various sections. She is a freshman at Pauli Murray College and plans to major in STEM and Humanities.

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