Protocol for genome-scale CRISPR screening in engineered lineage reporter hPSCs to study cell fate determination

This protocol describes a method for genome-scale CRISPR screening in genetically engineered human pluripotent stem cells (hPSCs) to study cell fate determination. It utilizes the germ layer reporter PAX6 and an inducible CRISPR/Cas9 knockout system. The protocol enables the identification of lineage-specific preventing genes. By knocking out genes in hPSCs and observing the resulting differentiation, one can identify genes that prevent neuroectoderm (nEc) or mesendoderm (ME) differentiation. The engineered hPSCs contain a PAX6 reporter and an inducible Cas9 system, allowing for controlled gene knockout and observation of PAX6 expression, a marker for human neuroectoderm cell fate.

• 1
  The protocol allows for the identification of genes that prevent lineage specification, termed Lineage Specification Preventing Genes (LPGs).
• 2
  CRISPR knockout screening in PAX6-tdTomato/iCas9 hPSCs allows unraveling LPGs which maintain pluripotency by preventing tri-lineage specification.
• 3
  Identified LPGs and their functional modules can provide insights into the genetic wiring and biological processes controlling hPSC self-renewal and tri-lineage specification.