Genetic Eraser

The new technology precisely and quickly destroys the target proteins

Anna Yudina, "Scientific Russia"

Now researchers can more precisely target certain proteins in yeast, mammalian and mouse cells to study how a violation of certain protein properties can affect physical manifestation in a cell or an organism, writes eurekalert.org with reference to Nature Communications (Yesbolatova et al., The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice – VM).

"Conditional gene shutdown and small interfering RNA (miRNA), which is used to suppress proteins without completely disabling them, have been used in many studies," said Masato T. Kanemaki, professor at the National Institute of Genetics at the Research Organization of Information and Systems. "However, these technologies are not ideal for studying highly dynamic processes, such as the cell cycle, differentiation, or neural activity, due to the slow rate of depletion of the protein of interest."

Kanemaki and his team have previously developed an approach called the AID system, which uses a small protein label known as a degron fused with proteins to cause degradation. To start the decomposition process, the researchers introduced auxin, a plant hormone that helps regulate plant growth. In previous studies of conditional gene knockout and siRNA, according to Kanemaki, it usually takes two or three days to deplete the target protein. The AID system allows for a general, more efficient approach in which the target protein can be depleted in less than a few hours.

"The initial AID system has two major drawbacks: loose decomposition and the need for a high dose of auxin," Kanemaki said. "These negative features make it difficult to accurately control the expression level of the protein of interest in living cells and to apply this method in mice."

Now researchers can more precisely target certain proteins in yeast, mammalian and mouse cells to study how a violation of certain protein properties can affect physical expression in a cell or an organism.

The ability to knock out genes in mice is a critical step in genetic research and therapy. According to Kanemaki, this approach may work well in cultured cells, but it should work in the entire model system – in the entire body, for example, mice. The "loose degradation" of the AID system means that the target protein will only decompose weakly without auxin, but the level of auxin required to induce complete degradation appears to have a long-term negative effect on cell growth.

"In this paper, we describe an AID2 system that overcomes all the drawbacks of the original AID system," Kanemaki said, noting that they did not detect decomposition leaks in the system, decomposition was faster, and the required dose of auxin was much lower.

To create the AID2 system, the researchers used a so-called "bulge and hole" strategy to create an empty space in a mutant version of a plant protein (called TIR1) that recognizes and causes degradation of proteins fused with degron. The auxin analog can bind directly to the TIR1 mutant and initiate the degradation process. Since this approach is very effective, less auxin analog is required. The researchers found that depletion can be caused by a concentration about 670 times lower than in the original system.

"With the AID 2 system, it is possible to quickly deplete the protein of interest in cultured cells and mice,– Kanemaki said. "Then we plan to use this system to find something new in chromosome biology and apply the AID2 system to other model organisms."

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