Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells Journal Article
| Authors: | Wu, Y.; Zhong, A.; Evangelisti, A.; Sidharta, M.; Huangfu, D.; Studer, L.; Zhou, T. |
| Article Title: | Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells |
| Abstract: | Verification of genome editing in human pluripotent stem cells (hPSCs), particularly at silent loci, is desirable but challenging, as it often requires complex and time-intensive differentiation to induce their expression. Here, we establish a rapid and effective workflow for verifying genome-edited hPSC lines targeting unexpressed genes using CRISPR-mediated transcriptional activation (CRISPRa). We systematically compared the efficiency of various CRISPRa systems and identified the synergistic activation mediator (SAM) system as the most potent for activating silent genes in hPSCs. Furthermore, combining SAM with TET1, a demethylation module, enhanced the activation of methylated genes. By inducing targeted gene activation in undifferentiated hPSCs using CRISPRa, we successfully verified single- and dual-reporter lines, functionally tested degradation tag (dTAG) knockins, and validated silent gene knockouts within 48 h. This approach bypasses the need to induce target gene expression through differentiation, providing a rapid and effective assay for verifying silent gene editing at the hPSC stage. © 2025 The Author(s) |
| Keywords: | human cell; genetics; cytology; metabolism; gene; gene expression; transcription initiation; cell line; cell differentiation; dna methylation; gene activation; reporter gene; pluripotent stem cell; pluripotent stem cells; transcriptional activation; demethylation; procedures; pharmaceutics; human pluripotent stem cells; humans; human; article; clustered regularly interspaced short palindromic repeat; crispr cas system; gene editing; crispr-cas systems; gene knockout; silent gene; clustered regularly interspaced short palindromic repeats; transactivation assay; knockouts; crispr-mediated transcriptional activation; dtag system; knockins; reporter lines; silent gene editing |
| Journal Title: | Stem Cell Reports |
| Volume: | 20 |
| Issue: | 6 |
| ISSN: | 2213-6711 |
| Publisher: | Cell Press |
| Date Published: | 2025-06-10 |
| Start Page: | 102499 |
| Language: | English |
| DOI: | 10.1016/j.stemcr.2025.102499 |
| PUBMED: | 40345204 |
| PROVIDER: | scopus |
| PMCID: | PMC12181958 |
| DOI/URL: | |
| Notes: | The MSK Cancer Center Support Grant (P30 CA008748) is acknowledged in the PDF. Corresponding MSK authors are Lorenz Studer and Ting Zhou -- Source: Scopus |
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