Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research

Abstract

Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock‐down, knock‐out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR‐Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA‐based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in‐depth information, we will provide specific references throughout.

FEBS fellowships: supporting excellent science for over four decades

“…The second Mini‐review takes us from the molecular to the cellular level, as FEBS fellow Verónica Miguel together with Rafael Kramann describes metabolic reprogramming in renal fibrosis, a field where omics technology, single cell/nucleus RNA sequencing, and advances in software are facilitating the capture of metabolic profiles at unparalleled spatial and temporal resolution [ 2 ]. The third article, by FEBS fellow Stefano L. Giandomenico together with Erin M. Schuman, provides a broad overview of the technologies available to study gene function in mammals, including the use of CRISPR and targeted protein degradation [ 3 ]. This article provides a fantastic introduction for those new to the field, and is a useful central hub for obtaining full details on this wide range of methodologies.…”