Targeting protein function: the expanding toolkit for conditional disruption

Abstract

A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

“…Gene products that are essential for cell survival can be conditionally depleted by a number of methods at DNA, RNA or protein level 22 . In order to provide SORTS with this capability, we took advantage of an auxin-inducible degron (AID) from plant transcriptional repressor IAA17 and rice transport inhibitor response 1 (osTIR1) protein.…”

Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation

“…56 Using a reporter with reduced half-lifea commonly used approach to study dynamic processes in mammalian cells 56,57 partly alleviates this issue, but also results in loss dynamic range and sensitivity of the output signal. 56,60 We demonstrated here that integrating the NanoDeg system into a genetic circuit to achieve stimulus-dependent degradation of the reporter mediated by the NanoDeg allows recapitulating the input dynamic behavior with enhanced accuracy.…”

“…11−15 While providing valuable tools to enable inducible and tunable control of target degradation, [10][11][12][13][14][15]59 they require genetic manipulation of the target protein, which may affect the target protein's native function. 60 Heterobifunctional molecules, such as PROTACs 17−21 and the Ubiquibodies, 22 consisting of fusion of UPS-and target-specific recognition elements, induce interaction between the proteasome and the target protein, thus mediating target depletion without the need for target manipulation. 22−25 These methods, however, are typically target-specific, have not been implemented to generate a universal technology for targeting degradation of endogenous proteins, and do not allow tunable control over the extent of target depletion.…”

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

“…To date, the suppression of a specific gene function by gene silencing or editing has been successfully performed in many organisms (5,6). In addition genetically manipulated model organisms with cytotoxic protein-induced dysfunction of specific tissues would be crucial for the study of various biological applications and mechanisms.…”