[PDF][PDF] Scarless genome editing of human pluripotent stem cells via transient puromycin selection

B Steyer, Q Bu, E Cory, K Jiang, S Duong, D Sinha… - Stem cell reports, 2018 - cell.com
B Steyer, Q Bu, E Cory, K Jiang, S Duong, D Sinha, S Steltzer, D Gamm, Q Chang, K Saha
Stem cell reports, 2018cell.com
Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease
modeling, drug discovery, and regenerative medicine. We present and characterize a robust
method for rapid, scarless introduction or correction of disease-associated variants in hPSCs
using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-
acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9,
we transiently select for cells based on their early levels of Cas9 protein. Under optimized …
Summary
Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.
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