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Summary Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells., eLife digest Genes are often referred to as the blueprints of life. Understanding the role of the genes in human cells is one of the major goals of biology. Recent advances in gene editing technologies, such as CRISPR/Cas9, mean scientists can now edit or delete precise sections within human genes, similar to how we edit words in a document on a computer. This has made it possible to insert small sequences that encode specific “tags” into genes. This in turn means that when a protein is built following the instructions in the gene, the protein includes the tag too, making it easy to monitor. Tags on proteins can help scientists understand what those proteins do by answering various questions, such as: where is the protein found in the cell? How much of the protein is there in each cell? Does this change as the cell matures? What does the protein interact with? Yet, more research could be done if the tagging process was made easier, quicker and more efficient. Dewari et al. have now come up with an improved gene editing approach that enabled them to rapidly tag hundreds of proteins all at the same time, with efficiencies that were much higher than expected based on previous approaches. The strategy uses common “off the shelf” reagents that can be designed with a new user-friendly, web-based tool called “Tag-IN”. Dewari et al. focused on optimizing their method in freshly grown stem cells, originally collected from mice and humans. They then went on to show the scalability and efficiency of this approach by tagging 60 different proteins in brain stem cells from mice. Now, rather than being limited to a handful of genes of interest, scientists can explore large families of genes in a variety of mouse and human cells in a much quicker and more comprehensive manner. Also, working with stem cells that can be freshly collected from individuals rather than cells that have been grown in the laboratory for a long time will be more useful for biological and disease studies. In the long-term, more knowledge of how protein-coding genes work in different human cells will benefit patients as new drugs or therapeutic targets are discovered.