Jun 16, 2026

CRISPRa Screening for PDX1 CCR in Human Embryonic Stem Cells

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvojx7xg4o/v1
  • 1Sloan Kettering Institute;
  • 2Johns Hopkins University
Gary Yang
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvojx7xg4o/v1
Protocol Citation: Julian Pulecio, Michael Beer, Danwei Huangfu 2026. CRISPRa Screening for PDX1 CCR in Human Embryonic Stem Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvojx7xg4o/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 30, 2026
Last Modified: June 16, 2026
Protocol  Integer ID: 318240
Keywords: CRISPRa, PDX1, CRISPR Screen, Competent Chromatin Region, Human Embryonic Stem Cell, crispra screening for pdx1 ccr, crispr activation, crispra screening, human embryonic stem cell, embryonic stem cell, pdx1 gene, competent chromatin region, pdx1 ccr, screening approach in human, screening approach
Funders Acknowledgements:
NHGRI
Grant ID: U01 HG012051
Abstract
To identify and validate competent chromatin regions (CCRs) of the PDX1 gene using a CRISPR activation (CRISPRa) screening approach in human embryonic stem cells (hESCs).
CRISPRa Screening for PDX1 CCR in Human Embryonic Stem Cells
The PDX1 lentiviral library with the MS2-gRNA backbone was transduced into 12 million hESC iSAM cells distributed in 10 x 10 cm plates, while the library cloned using the regular lenti-gRNA backbone was transduced into 12 million hESC dCas9-VP64 cells distributed in 10 x 10 cm plates, to reach an MOI =0.3. Cells were reverse transduced in the presence of RI (10 μM) and PS (6 μg/ml).
Both transduced cell lines were kept under puromycin selection (1 μg/ml) for 4 days, and after one day of recovery with regular E8 media, were grown with Dox during four additional days.
The presence of cells expressing PDX1 (reported by GFP) was verified by flow cytometry, after being tained with LIVE/DEAD reagent (Thermo Fisher Scientific, L34955) for 15 mins at RT.
All the iSAM-hESCs expressing PDX1 (GFP+) cells were FACS-sorted using FACSaria sorters (BD Biosciences) by the MSKCC cytometry facility; a corresponding number of PDX1-GFP (-) cells were also sorted, 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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