(41) Programmable Induction of Human Peri-Implantation Embryonic Lineages by CRISPR Activation

Carly Guiltinan^1,2,3; Clara J. Han^1,2,3; Gerrald A. Lodewijk^1,2,3; Benjamin R. Topacio^1,2,3; S. Ali Shariati^1,2,3

1. Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA

2. Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA

3. Institute for the Biology of Stem Cells, University of California, Santa Cruz, Santa Cruz, CA, USA

Abstract Text:

Successful pregnancy depends on the proper formation of two major embryonic lineages: (1) trophoblast, which becomes the fetal placenta, and (2) hypoblast, which forms the yolk sac and provides instructive signals for embryonic development. These structures, collectively known as extraembryonic tissues, are essential for implantation, which is one of the most vulnerable times for pregnancy loss. Despite their importance, our understanding of the transcriptional regulatory networks that specify human extraembryonic cells remains limited. This gap in knowledge primarily results from the scarcity of human embryos to systematically study human-specific transcriptional regulatory networks that specify embryonic cell fates. CRISPR-based activation (CRISPRa) can uncover the minimal epigenetic processes required for specification of human hypoblast and trophoblast lineages via activation of endogenous regulatory elements of cell fate-determining factors. Our central hypothesis is that intrinsic activation of endogenous regulatory elements of fate-determining transcription factors will overcome the epigenetic barriers required for cell fate transitions, thereby inducing the transcriptional programs necessary for the efficient differentiation of hypoblast and trophoblast cells. To test this hypothesis, we first established dox-inducible dCas12a-based CRISPRa human embryonic stem cell (hESC) lines using a non-viral piggyBac transposon system. Uniquely, dCas12a allows for straightforward multiplexing by processing arrays of gRNAs targeting different regulatory elements separated by repeated elements. Using existing single-cell transcriptomics data of natural human embryos, two gRNA arrays were tested for each extraembryonic lineage to activate major cell fate-determining transcription factors: GRHL1 + GRHL3 and TFAP2A + TFAP2C + GATA2 + GATA3 for trophoblast, and GATA6 and GATA4 + FOXA2 + SOX17 for hypoblast, along with a non-targeting gRNA for LacZ as a control. Non-dox-treated wells for each gRNA candidate were used as additional controls to assess induction efficiency. Quantitative gene expression analysis demonstrated that dCas12a-based CRISPR activation can efficiently induce expression of multiple hypoblast and trophoblast transcription factors. Importantly, we observed induction of downstream lineage-specific factors that were not directly targeted by CRISPRa, indicating activation of transcriptional programs for the respective cell types in dox-treated cells compared to no dox controls. Single-cell immunostaining confirmed our gene expression findings by showing protein expression of targeted transcription factors, which was accompanied by a proportional downregulation of pluripotency (OCT4), though the extent of this response varied across individual cells. These findings suggest that dCas12a-based CRISPRa can convert hESCs to extraembryonic lineages through intrinsic epigenome editing and without relying on cell-specific morphogens. In ongoing experiments, we are assembling inducible hESC lines for each of the embryonic lineages to generate programmable models of human embryogenesis. This system will enable mechanistic studies of human development through precise CRISPR-based gene expression manipulation, similar to what our laboratory has recently established for mouse embryo models. Financial support: CIRM Postdoctoral fellowship and UCSC IBSC.