Title: Scarless genome editing of human stem cells via transient puromycin selection
Legend: Transient puromycin selection enriches for precise homology directed repair (HDR)-mediated genome editing in human pluripotent stem cells (hPSCs). hPSCs were electroporated to deliver CRISPR plasmids encoding both sgRNA and Cas9 with or without donor DNA (ssODN). Electroporated hPSCs were treated with puromycin from 24 to 96 hr (72 hr total) to select for cells with high transient expression of Cas9 from the non-integrated CRISPR plasmid. With this method we generated indel frequencies as high as 57%, HDR modified clones at rates as high as 36%, and isolation of scarless homozygous HDR edited clones at up to 13% efficiency. Cells edited using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation.
Citation: Steyer, B., Bu, Q., Cory, E., Jiang, K., Duong, S., Sinha, D., Steltzer, S., Gamm, D., Chang, Q., and Saha, K. (2018). Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection. Stem Cell Reports 10, 642–654.
Abstract: 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.
About the Lab: The Saha Lab is affiliated with several multi-disciplinary centers including the Waisman Center, Wisconsin Institute for Discovery and the Stem Cell and Regenerative Medicine Center at UW-Madison. Our research dedicated to using human stem cells together with emerging engineering methods in material science and synthetic biology to make smarter therapeutics, model human disease, and advance personalized medicine. As a part of our effort to develop new prognosis and diagnosis tools together with Audra Sterling, Jan Greenberg and Marsha Mailick of the Waisman Center, we investigate methods that can improve our understanding of the genotype-phenotype correlation between various genes and the outcomes such as linguistic and cognitive phenotypes.