CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9 & Jmjd6 , Kat6a & Jmjd6 , and Brpf1 & Jmjd6 in leukemia cells.

Barrett's esophagus is a common type of metaplasia and a precursor of esophageal adenocarcinoma. However, the cell states and lineage connections underlying the origin, maintenance, and progression of Barrett's esophagus have not been resolved in humans. To address this, we performed single-cell lineage tracing and transcriptional profiling of patient cells isolated from metaplastic and healthy tissue. Our analysis revealed discrete lineages in Barrett's esophagus, normal esophagus, and gastric cardia. Transitional basal progenitor cells of the gastroesophageal junction were unexpectedly related to both esophagus and gastric cardia cells. Barrett's esophagus was polyclonal, with lineages that contained all progenitor and differentiated cell types. In contrast, precancerous dysplastic foci were initiated by the expansion of a single molecularly aberrant Barrett's esophagus clone. Together, these findings provide a comprehensive view of the cell dynamics of Barrett's esophagus, linking cell states along the full disease trajectory, from its origin to cancer.

The growth potential of individual epithelial cells is a key determinant of tissue development, homeostasis, and disease progression. Although it is known that epithelial progenitor cells vary in their proliferative capacity, the cell states underlying these differences are yet to be uncovered. Here we performed clonal tracing through imaging and cellular barcoding of an in vitro model of esophageal epithelial cells (EPC2-hTERT). We found that individual clones possess unique growth and differentiation capacities, with a subset of clones growing exponentially. Further, we discovered that this proliferative potential for a clone is heritable through cell division and can be influenced by extrinsic cues from neighboring cells. Combining barcoding with single-cell RNA-sequencing (scRNA-seq), we identified the cellular states associated with the highly proliferative clones, which include genes in the WNT and PI3K pathways. Importantly, we also identified a subset of cells resembling the highly proliferative cell state in the healthy human esophageal epithelium and, to a greater extent, in esophageal squamous cell carcinoma (ESCC). These findings highlight the physiological relevance of our cell line model, providing insights into the behavior of esophageal epithelial cells during homeostasis and disease.