Coupling a developmental promoter to CRISPR interference for Wnt pathway regulation in human pluripotent stem cells
- 1University of Wisconsin - Madison
Abstract
While directed differentiation of human pluripotent stem cells commonly relies on the timed delivery of extracellular factors, these uniform treatments often yield heterogeneous responses across cell populations. Linking intracellular gene regulation directly to an emerging developmental state offers a complementary strategy to coordinate these differentiation signals from within the cell. Here, we explored this approach by coupling a T/Brachyury promoter to CRISPR interference targeting CTNNB1, which encodes the canonical Wnt signaling mediator {beta}-catenin. A T-promoter EGFP reporter line exhibited transiently increased activity during early differentiation, supporting the use of this promoter as a developmentally responsive input. We then combined the promoter with dCas9-KRAB and a CTNNB1-targeting guide RNA. Cas9-mediated integration was accompanied by indels at the CTNNB1 target site, whereas a Cas12a-mediated integration strategy yielded clones with no indels detected by ICE analysis. During differentiation, the selected T-dCas9 CTNNB1 clone showed reduced CTNNB1 expression and attenuated induction of Wnt associated genes. Together, these findings provide a proof of concept for combining a developmental promoter with a programmable intracellular regulator and identify a strategy for separating circuit integration from unintended target-site editing. This modular approach provides a foundation for developing genetic interventions whose expression is linked to developmental state.
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BibTeX
@misc{jin2026coupling,
title = {{Coupling a developmental promoter to CRISPR interference for Wnt pathway regulation in human pluripotent stem cells}},
author = {G. Jin and S. P. Palecek},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.25.754435},
url = {https://www.biorxiv.org/content/10.64898/2026.09.25.754435v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Coupling a developmental promoter to CRISPR interference for Wnt pathway regulation in human pluripotent stem cells
AU - G. Jin
AU - S. P. Palecek
PY - 2026
DA - 2026/09/25
PB - bioRxiv
DO - 10.64898/2026.09.25.754435
UR - https://www.biorxiv.org/content/10.64898/2026.09.25.754435v1
AB - While directed differentiation of human pluripotent stem cells commonly relies on the timed delivery of extracellular factors, these uniform treatments often yield heterogeneous responses across cell populations. Linking intracellular gene regulation directly to an emerging developmental state offers a complementary strategy to coordinate these differentiation signals from within the cell. Here, we explored this approach by coupling a T/Brachyury promoter to CRISPR interference targeting CTNNB1, which encodes the canonical Wnt signaling mediator {beta}-catenin. A T-promoter EGFP reporter line exhibited transiently increased activity during early differentiation, supporting the use of this promoter as a developmentally responsive input. We then combined the promoter with dCas9-KRAB and a CTNNB1-targeting guide RNA. Cas9-mediated integration was accompanied by indels at the CTNNB1 target site, whereas a Cas12a-mediated integration strategy yielded clones with no indels detected by ICE analysis. During differentiation, the selected T-dCas9 CTNNB1 clone showed reduced CTNNB1 expression and attenuated induction of Wnt associated genes. Together, these findings provide a proof of concept for combining a developmental promoter with a programmable intracellular regulator and identify a strategy for separating circuit integration from unintended target-site editing. This modular approach provides a foundation for developing genetic interventions whose expression is linked to developmental state.
ER -