Background

Knockout or overexpression screens are efficient methods for identifying the involvement of novel genes that contribute to phenotypes such as drug resistance. Perturbation of gene function is enabled through either loss-of-function studies, using biological tools such as the CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats) (Humphrey and Kasinski, 2015; Szlachta et al., 2018; Li and Kasinski, 2020), or by gain-of-function studies through overexpression of human ORFs (Open Reading Frame) (Arnoldo et al., 2014). Several genes can potentially be identified by such screening methodologies; however, validation is a key step. To validate and dissect the cellular function of the gene(s) of interest, gene modulation has become a prevalent technique in the field. To this end, the candidate gene is either individually knocked out or overexpressed, single clones are isolated, and finally the phenotype observed via the screen is reevaluated. Nevertheless, to confirm that the gene of interest is accurately knocked out or overexpressed in single clones, protein quantification is a usual process.

Differential protein expression of individual clones is conventionally determined by western blot. This and other techniques such as immunofluorescence and immunohistochemistry are indispensable methods for protein analysis; however, these protocols require increased amounts of resources (antibodies, reagents) and often need one or more days to complete. Therefore, a semi-high-throughput screen that allows for rapid identification of differential protein expression post-clonal selection that reduces expenses, labor, and time should be considered. The in-cell western (ICW) is a powerful, simple, and reproducible technique that is underutilized in the field. It is a cost-effective method to quantify intracellular signaling in intact cells. The ICW protocol involves fixation and immunostaining of cells and combines the specificity of a western blot with the reproducibility and throughput of an enzyme-linked immunosorbent assay (ELISA) (Boveia and Schutz-Geschwender, 2015). From previous reports, semi-high-throughput cell-based applications of ICW include: 1) identification of efficient siRNAs from libraries and 2) identification of small molecule inhibitors targeting a particular signaling pathway (Hoffman et al., 2010). Additionally, ICW has been successfully utilized for screening genotoxic drugs by quantifying the expression of γH2AX, a well-known DNA damage and repair marker (Khoury et al., 2013). Another example of the versatility of ICW for throughput experiments is during the screening of chemical libraries for compounds that modulate the intensity and duration of growth factor-induced MAPK activity, an important regulator in cancer progression (Schnaiter et al., 2014).

Here, we describe a semi-high-throughput screening mechanism using the ICW protocol for validation of single knockout or overexpression clones for a protein of interest, initially identified using the CRISPR-Cas9 screening system (Pal et al., 2020, 2022). ICW has proved to be an efficient technique for clonal selection of cells because it allows rapid analysis of numerous samples, conserving the accuracy of the quantifiable output. To the best of our knowledge, the ICW protocol described below is the first reported use of ICW for the selection of multiple single clones simultaneously.

The pros and cons of using ICW versus western blot for efficient clonal selection of cells are enlisted below (Table 1).

Table 1. Pros and cons of using in-cell western over using the more conventional technique of western blot for high-throughput selection of single clones.

The protocol described here is validated for one protein; therefore, optimization of various parameters may be necessary to achieve study-specific goals, described in Table 2.

Table 2. Parameters at specific steps of ICW that can be optimized to achieve high-throughput selection of single clones for study-specific goals.