Mapping the Temporal Impact of Perturbations on Lineage Plasticity and Gene Regulation

This study characterizes gene and enhancer knockout phenotypes of 30 genes in a human pluripotent stem cell (hPSC)-directed islet differentiation system. Samples were collected at five differentiation stages from hPSCs to SC-islet cells for scRNA-seq to examine transcriptional impact of the knockouts over time. 30 genes were edited using CRISPR-Cas9, including HHEX, GATA6, PDX1, GATA4, FOXA1, FOXA2, MNX1, NEUROD1, NKX2-2, RFX6, GSC, HNF4A, PAX6, PBX1, GLIS3, NEUROG3, ARX, OTUD5, BMPR1A, TLE3, BCOR, QSER1 , TET1, TET2, TET3, TADA2B, KDM2B, PROSER1 gene knockouts, and HHEX, ONECUT1 and NANOG enhancer deletions. The CRISPR-Cas9 edited lines were generated in two hPSC backgrounds (HUES8 and H1 human embryonic stem cells), with each genotype represented by multiple clones. The clonal lines were individually barcoded (using the expressed LARRY barcodes) and pooled for differentiation. Samples were collected at five differentiation stages as follows, ESC, definitive endoderm (DE, Day 3), Posterior foregut (PFG or PP1, Day 7), pancreatic progenitor (PP, Day 11), islet-like (SC-islet, Day 18) for scRNA-seq. In addition, WT cells in H1 and HUES8 backgrounds were differentiated and sequenced in parallel at 17 time points: day -1 (ESC), 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 21, 25, 26, 35. Please note that processed .h5ad data was generated from both *transcriptome and *larry samples, and is linked to the corresponding *transcriptome records.