Background

The epidermal growth factor (EGF) receptor (EGFR) is activated by EGF or other ligands at the cell surface, which triggers the RAS-RAF-MAPK/ERK1/2 signaling pathway involved in cell proliferation, differentiation, survival, and motility (Karnoub and Weinberg, 2008). Activated EGFR is endocytosed and accumulated in early endosomes, where it is either recycled back to the plasma membrane or targeted for lysosomal degradation. Whether active EGFR continues to signal through the RAS-ERK1/2 axis from endosomes to sustain ERK1/2 activation remains controversial and is under debate in the literature (reviewed in von Zastrow and Sorkin, 2021). Evidence for the endosomal signaling to ERK1/2 is based on the detection of components of the ERK1/2 pathway in endosomes in cells stimulated with EGF (Pol et al., 1998; Howe et al., 2001; Jiang and Sorkin, 2002; Lu et al., 2009; Schmick et al., 2014). However, these observations were made either with overexpressed recombinant proteins or with chemically fixed cells or by subcellular fractionation, approaches that may not correctly report subcellular localization of endogenous proteins. We initially studied the spatio-temporal dynamics of the endogenous tagged EGFR and components of the ERK1/2 pathway by fluorescently tagging these proteins using gene editing techniques utilizing zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) (Pinilla-Macua et al., 2017, 2016). Subsequently, we switched to using the CRISPR-Cas9 system (Surve et al., 2019; Surve et al., 2021) for gene-editing. We selected for these studies a subvariant of HeLa cells that we consistently used in the laboratory because they express EGFR at levels similar to those in many mammalian cells in vivo, and because we have rigorously characterized the EGFR endocytic trafficking system in these cells.

CRISPR-Cas9 has proven to be versatile and efficient method of gene editing compared to ZFNs and TALEN, and is widely used for gene knockouts, gene activation, gene inhibition, and knock-ins. The fascinating discovery and evolution of CRISPR technology is beyond scope of this article, and excellent reviews on this topic can be found elsewhere. We used this technology to tag KRAS, NRAS, and Grb2. Generally, there are two main components of CRISPR-Cas9 technology: i) Cas9—a RNA guided endonuclease, ii) a short non-coding guide RNA consisting of a target complementary CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) that shuttles Cas9 to a specific site. These components are delivered inside the cells via plasmids or via direct delivery of an in vitro generated complex of purified Cas9 and purified sgRNA (Ran et al., 2013). Following the delivery, gRNA through Watson-Crick base pairing binds to its target DNA sequence, and Cas9 makes a double stranded break (DSB) near the PAM site of gRNA. The DSB is either repaired by the error prone non-homologous end joining (NHEJ) mechanism or by precise homology dependent repair (HDR) mechanism. In case of gene tagging, an additional component is provided in the form of a repair template, i.e., a donor DNA containing sequences of a fluorescent protein or small tags such as His, HA, or MYC, which through HDR allows the insertion of the tag of interest. HDR mechanism of repair is inherently inefficient, resulting in lower yields of clones of cells with the in-frame incorporated tag compared to generation of knockout clones, which generally involves NHEJ. The protocol here describes the variations (Table 1) in the approach by which gene tagging (knock-in) can be achieved in transformed cell lines like HeLa.