ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis (bulk RNAseq)

Bulk RNA sequencing was performed on MSK107Li patient-derived colorectal cancer (CRC) liver metastasis organoids to characterize transcriptional changes resulting from CRISPR-Cas9-mediated ZFP36L2 knockout during dedifferentiation. ZFP36L2 was disrupted using two independent sgRNAs; two validated knockout clones per sgRNA were pooled for sequencing, with control organoids serving as comparators. To assess dedifferentiation capacity, organoids were cultured in intestinal growth factor-free (IGFF) medium for 13 days to induce differentiation, then dissociated into single cells and passaged into HISC medium; samples were harvested on day 7 of dedifferentiation. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and submitted to Plasmidsaurus for 3′ end RNA-seq library preparation and sequencing. Libraries were generated using a 3′ end counting approach with unique molecular identifiers (UMIs) incorporated during cDNA synthesis. ZFP36L2 knockout organoids exhibited decreased ISC-associated gene signatures and increased expression of canonical and non-canonical differentiation programs, including osteoblast, squamous, and neuroendocrine lineages, relative to controls.