Tracing the transitions from pluripotency to germ cell fate with CRISPR screening

Early mammalian development entails a series of cell fate transitions that includes transit through naïve pluripotency to post-implantation epiblast. This can subsequently give rise to primordial germ cells (PGC), the founding population of the germline lineage. To investigate the gene regulatory networks that control these critical cell fate decisions, we developed a compound-reporter system to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with unbiased genome-wide CRISPR screening. This enabled identification of key genes both for exit from pluripotency and for acquisition of PGC fate, with further characterisation revealing a central role for the transcription regulators Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in significantly impaired PGCLC development accompanied with widespread activation (Nr5a2-/-) or inhibition (Zfp296-/-) of WNT pathway components. This leads to aberrant upregulation of the somatic programme or failure to appropriately activate germline genes in PGCLC, respectively, and consequently loss of germ cell identity. Overall our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate transitions. Sample1-36: mRNA-seq of epiblast-like cells (EpiLC), and PGCLC at day (D) 2 and D6 of differentiation; 3 biological independent replicates for following genotypes in a Stella-GFP; Esg1-tdTomato background: wild-type, Nr5a2-/-, or Zfp296-/-; Sample37-42: mRNA-seq of infected? embryonic stem cells (ESC), EpiLC and D6 PGCLC; 2 biological replicates; Sample 43-52: gRNA amplicon libraries from embryonic stem cells (ESC), EpiLC or D6 PGCLC from CRISPR screen; 2 biological independent replicates