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CRISPR Screen Highlights ZIC2 as an Essential Gene for Human Cardiogenesis

Review of “Genome-Wide CRISPR Screen Identifies ZIC2 As an Essential Gene That Controls the Cell-Fate of Early Mesodermal Precursors to Human Heart Progenitors” from STEM CELLS by Stuart P. Atkinson

A recent STEM CELLS article from the laboratory of Kenneth R. Chien (Karolinska Institutet, Stockholm, Sweden) sought to explore the genetic circuit responsible for cardiac progenitor cell formation, which represents one of the earliest steps of human cardiogenesis [1]. In this new study, Xu et al. employed a genome‐wide unbiased CRISPR‐knockout screen as a feasible and straightforward approach to the unbiased discovery of genes associated with a specific phenotype [2, 3]. Their findings now highlight the zinc-finger of the cerebellum 2 (ZIC2) gene [4, 5], a known master regulator of neurogenesis, as essential to the formation of human cardiac progenitor cells from embryonic stem cells (ESCs).

The authors employed the CRISPR‐knockout screen to assess the role of around 600 genes at two distinct stages during the differentiation of Cas9-expressing ESCs into cardiomyocytes – the formation of MESP1-expressing early cardiac mesoderm cells and ISL1-expressing multipotent cardiac progenitor cells. The screen output provided a list of fifteen candidate genes necessary for cardiac mesoderm (MAD2L1, PPP3CC, ZIC2, PODXL, NAPA, GPR4, and TTC39B) and cardiac progenitor cell formation (SEMA3B, MAGIX, TRUB2, SMAD4, FBXL19, CTSV, PLEKHO2, NOX3, and DFFA). After the validation of seven promising gene candidates (SMAD4, PLEKHO2, CTSV, TTC39B, ZIC2, SEMA3B, and MAGIX), the authors chose to study ZIC2 as a novel essential gene for human cardiac mesoderm and progenitor formation, as mutations to this gene prompted the most potent effect on cardiac differentiation efficiency.

To confirm the essential role of ZIC2, the authors created ZIC2-null ESCs; interestingly, these cells remained pluripotent, although they displayed a significantly reduced potential for the formation of cardiac progenitor cells from early mesodermal precursors. Instead, early mesodermal precursors derived from ZIC2-null ESCs displayed a preference for differentiation towards non-cardiac cell lineages (such as the osteoblast lineage) due to a lack of ZIC2-associated apelin receptor‐related signaling. Of note, apelin receptor signaling can modulate NODAL signaling, which represents a crucial mesodermal induction pathway [6].

Overall, these new findings forge a link between ZIC2 and human cardiogenesis and highlight the utility of genome‐wide unbiased CRISPR‐knockout screens in the exploration of developmental processes recapitulated by the directed differentiation of pluripotent stem cells.

For more on the genes, proteins, and pathways controlling human cardiogenesis, stay tuned to the Stem Cells Portal!


  1. Xu J, Zhou C, Foo KS, et al., Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors. STEM CELLS 2020;38:741-755.
  2. Shalem O, Sanjana NE, Hartenian E, et al., Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science 2014;343:84.
  3. Wang T, Wei JJ, Sabatini DM, et al., Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science 2014;343:80.
  4. Elms P, Siggers P, Napper D, et al., Zic2 is required for neural crest formation and hindbrain patterning during mouse development. Developmental Biology 2003;264:391-406.
  5. Houtmeyers R, Souopgui J, Tejpar S, et al., The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis. Cellular and Molecular Life Sciences 2013;70:3791-3811.
  6. Deshwar AR, Chng SC, Ho L, et al., The Apelin receptor enhances Nodal/TGFβ signaling to ensure proper cardiac development. Elife 2016;5:e13758.