AI-designed protein wakes up silent genes, one by one

By combining CRISPR technology with a protein engineered with artificial intelligence, it is possible to wake up individual dormant genes by turning off the chemical “kill switches” that silence them. Researchers at the University of Washington (UW) School of Medicine in Seattle describe this discovery in the magazine Cell reports.

The approach will allow researchers to understand the role that individual genes play in normal cell growth and development, in aging and in diseases such as cancer, said Shiri Levy, postdoctoral fellow at the UW Institute for Stem. Cell and Regenerative Medicine (ISCRM) and the main author of the article.

“The beauty of this approach is that we can safely upregulate specific genes to affect cellular activity without permanently altering the genome and causing unintended errors,” Levy said.

The project was led by Hannele Ruohola-Baker, professor of biochemistry and associate director of ISCRM. The AI-designed protein was developed at the UW Medicine Institute for Protein Design (IPD) under the direction of David Baker, also a professor of biochemistry and director of IPD.

The new technique monitors gene activity without altering the DNA sequence of the genome by targeting chemical changes that help package genes into our chromosomes and regulate their activity. Because these modifications do not occur in genes, but on top of genes, they are called epigenetics, from the Greek epi “above” or “above” the genes. Chemical changes that regulate gene activity are called epigenetic markers.

Scientists are particularly interested in epigenetic modifications because not only do they affect the activity of genes in normal cell function, but epigenetic markers accumulate over time, contribute to aging and can affect the health of future generations because we can pass them on to our children.

In their work, Levy and his colleagues focused on a complex of proteins called PRC2 that silences genes by attaching a small molecule, called a methyl group, to a protein that packages genes called histones. These methyl groups must be refreshed so that if PRC2 is blocked, the genes it has silenced can be awakened.

PRC2 is active throughout development but plays a particularly important role during the first days of life when embryonic cells differentiate into different cell types that will form the tissues and organs of the growing embryo. PRC2 can be blocked with chemicals, but they are imprecise, affecting PRC2 function throughout the genome. The goal of the UW researchers was to find a way to block PRC2 so that only one gene at a time was affected.

To do this, Baker and his colleagues used AI to create a protein that would bind to PRC2 and block a protein that PRC2 uses to modify histones. Ruohola-Baker and Levy then fused this engineered protein with a disabled version of a protein called Cas9.

Cas9 is the protein used in the gene editing process called CRISPR. Cas9 binds and uses RNA as an address tag. The system allows scientists, by synthesizing a specific “address tag” RNA, to bring Cas9 to a precise location in the genome and therefore cut and splice genes at specific sites. In this experiment, however, the cutting function of the Cas9 protein is disabled, so the genomic DNA sequence is not changed. Accordingly, it is called dCas9, for “death”. However, the Cas9 function as a “vehicle” to deliver cargo to a specific location remains active. The blocking protein designed by the AI ​​was the cargo of the dCas9-RNA construct. “dCas9 is like UBER,” says Levy, “it will take you anywhere on the genome you want to go. The RNA guide is like a passenger, telling the UBER where to go.

In the new paper, Levy and his colleagues show that using this technique they were able to block PRC2 and selectively turn on four different genes. They were also able to show that they could transdifferentiate induced pluripotent stem cells into placental progenitor cells by simply turning on two genes.

“This technique allows us to avoid bombarding cells with various growth factors and gene activators and repressors to get them to differentiate,” Levy said. “Instead, we can target specific sites on the gene transcription promoter region, lift those marks, and let the cell do the rest organically and holistically. “

Finally, the researchers were able to show how the technique can be used to find the location of specific regulatory regions controlled by PRC2 from which individual genes are activated. The location of many of them is unknown. In this case, they identified a promoter region – called the TATA box – for a gene called TBX18. Although the current thought is that these promoter regions are close to the gene, within 30 base pairs of DNA, they found that for this gene the promoter region was over 500 base pairs away.

“It was a very important finding,” Ruohola-Baker said. “TATA boxes are scattered throughout the genome, and current thinking in biology is that the important TATA boxes are very close to the gene transcription site and the others don’t seem to matter. The power of this tool is that it can find the critical PRC2 dependent items, in this case the TATA boxes that matter.

Epigenetic changes decorate large regions of the genome in normal and abnormal cells. However, the minimum functional unit for epigenetic modification remains poorly understood, notes Ruohola-Baker: “With these two advances, AI-engineered proteins and CRISPR technology, we can now find the precise epigenetic marks that are important for gene expression, learn the rules and use them to control cell function, drive cell differentiation and develop 21st century therapies.

– This press release was originally posted on the University of Washington Medical School website

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