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Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Reactive oxygen species (ROS) are important factors mediating aging according to the free radical theory of aging. Few studies have systematically measured ROS levels in relationship to aging, partly due to the lack of tools for detection of specific ROS in live animals. By using the H₂O₂-specific fluorescence probe Peroxy Orange 1, we assayed the H₂O₂ levels of live Caenorhabditis elegans with 41 aging-related genes being individually knocked down by RNAi. Knockdown of 14 genes extends the lifespan but increases H₂O₂ level or shortens the lifespan but decreases H₂O₂ level, contradicting the free radical theory of aging. Strikingly, a significant inverse correlation between lifespan and the normalized standard deviation of H₂O₂ levels was observed (p < 0.0001). Such inverse correlation was also observed in worms cultured under heat shock conditions. An oxidative fluctuation hypothesis of aging is thus proposed and suggests that the ability of animals to homeostatically maintain the ROS levels within a narrow range is more important for lifespan extension than just minimizing the ROS levels though the latter still being crucial.

Reactive oxygen species (ROS) are important factors mediating aging according to the free radical theory of aging. Few studies have systematically measured ROS levels in relationship to aging, partly due to the lack of tools for detection of specific ROS in live animals. By using the H₂O₂-specific fluorescence probe Peroxy Orange 1, we assayed the H₂O₂ levels of live Caenorhabditis elegans with 41 aging-related genes being individually knocked down by RNAi. Knockdown of 14 genes extends the lifespan but increases H₂O₂ level or shortens the lifespan but decreases H₂O₂ level, contradicting the free radical theory of aging. Strikingly, a significant inverse correlation between lifespan and the normalized standard deviation of H₂O₂ levels was observed (p < 0.0001). Such inverse correlation was also observed in worms cultured under heat shock conditions. An oxidative fluctuation hypothesis of aging is thus proposed and suggests that the ability of animals to homeostatically maintain the ROS levels within a narrow range is more important for lifespan extension than just minimizing the ROS levels though the latter still being crucial.