Dissecting human endoderm development through a genome-wide CRISPR screen in human embryonic stem cells

Combining human ES cell differentiation with the CRISPR/Cas technology, we have conducted a genome-wide knockout screen for genes that cell-autonomously regulate the ?formation of human definitive endoderm (DE), which gives rise to most of the cells in respiratory ?and gastrointestinal organs including the lung, pancreas and liver. Our screen identified ?previously known as well as unknown regulators of endoderm differentiation. We further report ?that the MEKK1-MKK4/7-JNK1-JUN signaling axis acts as a previously unrecognized inhibitory ?pathway that constrains endoderm formation. ? Overall design: Genomic DNA from sorted SOX17GFP+/- cells were extracted by using Qiagen Maxi Kit (Qiagen 13362). Two-step PCR method was used to amplify the gRNA sequence and prepare Hi-seq library.