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 in silent locus is desirable but challenging because it often requires complex and time-intensive lineage-specific or tissue-specific differentiation to induce their expression. Here, we establish a rapid and effective workflow for the verification of hPSC lines with genome editing in unexpressed genes using CRISPR-mediated transcriptional activation (CRISPRa). We systematically compared the efficiency of various CRISPRa systems in hPSCs, identifying the SAM system as the most potent for activating silent genes in hPSCs. Furthermore, we demonstrated enhanced gene activation by combining the SAM system with TET1, a demethylation module. By inducing targeted gene activation in undifferentiated hPSCs using CRISPRa, we successfully verified single and dual reporter hPSC lines and conducted functional tests of dTAG knock-ins and silent gene knockouts within 48 hours. This approach eliminates the need for cell differentiation to access genes only expressed by differentiated cells, offering a handy assay for verifying gene editing in hPSCs.