Jun 17, 2026
CRISPRi Screening for PDX1 Enhancers in Pancreatic Progenitor Cells
- Julian Pulecio1,
- Michael Beer2,
- Danwei Huangfu1
- 1Sloan Kettering Institute;
- 2Johns Hopkins University
Protocol Citation: Julian Pulecio, Michael Beer, Danwei Huangfu 2026. CRISPRi Screening for PDX1 Enhancers in Pancreatic Progenitor Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmdow6v3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 31, 2026
Last Modified: June 17, 2026
Protocol Integer ID: 318236
Keywords: CRISPRi, PDX1, Human Embryonic Stem Cell, FACS Screen, Enhancer, pdx1 enhancers in pancreatic progenitor cell, pancreatic progenitor cell, crispr interference, enhancers of the pdx1 gene, human embryonic stem cell, pdx1 gene, pdx1 enhancer, embryonic stem cell, enhancer, crispri, screening approach in human
Funders Acknowledgements:
NHGRI
Grant ID: U01 HG012051
Abstract
To identify and validate enhancers of the PDX1 gene using a CRISPR interference (CRISPRi) screening approach in human embryonic stem cells (hESCs) differentiated to pancreatic progenitor cells.
CRISPRi Screening for PDX1 Enhancers in Pancreatic Progenitor Cells
The PDX1 lentiviral library with the regular lenti-gRNA backbone was reverse transduced into 12 million hESC (i)dCas9-KRAB hESCs distributed in 10 x 10 cm plates to reach a MOI =0.3. Cells were reverse transduced in the presence of RI (10 μM) and PS (6 μg/ml).
Cells were kept under puromycin selection for 4 days, and after one day of recovery with regular E8 media.
12 million selected cells were seeded into 6-well plates (4x10^5 per well) in VTN-coated plates, grown for 48 hours in E8 medium with Dox.
After washing with Phosphate Buffered Saline (PBS) w/o Ca2+ & Mg2+, cells were differentiated into Pancreatic Progenitor (PP) cells utilizing a protocol described previously (see hESC differentiation below).
Rezania, A., Bruin, J.E., Arora, P., Rubin, A., Batushansky, I., Asadi, A., O'Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T., et al. (2014). Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133. 10.1038/nbt.3033.
Doxycycline treatment was administered throughout the nine-days differentiation process.
After, cells were stained with LIVE/DEAD reagent and FACS-sorted using FACSaria sorters (BD Biosciences) based on PDX1 expression (reported by GFP expression) by the MSKCC cytometry facility.
PDX1-positive and PDX1-negative cells were sorted, aiming for a minimum representation = 500x (number of cells with a unique sgRNA).
Cells were pelleted down and kept at -80 for downstream gRNA sequencing. The screen was performed twice using independent biological replicates.
Protocol references
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