Highly efficient generation of isogenic pluripotent stem cell models using prime editing
- University of California, Berkeley, United States
- Albert Einstein College of Medicine, United States
- University of California, San Francisco, United States
Abstract
The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multi-component editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair (HDR) of site-specific nuclease-induced double-strand breaks (DSBs). Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs (ngRNAs) improved editing efficiencies up to 13-fold compared to transfecting the prime editing components as plasmids or ribonucleoprotein particles (RNPs). Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
Data availability
Sequencing data can be accessed through the repository platform Zenodo (10.5281/zenodo.6941502). The datasets for AAVS1 knock-in genotyping, aCGH karyotyping, and the source data files used to generate the featured graphs and tables can be found on Zenodo (10.5281/zenodo.6941599). Plasmids referred to in this paper have been deposited to Addgene's Michael J. Fox Foundation Plasmid Resource and their associated RRID can be found in Supplemental table 2.
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Highly efficient generation of isogenic pluripotent stem cell models using prime editing - DatasetsZenodo, doi:10.5281/zenodo.6941599.
Article and author information
Author details
Funding
Aligning Science Across Parkinson's (ASAP-000486)
- Luke A Gilbert
- Helen S Bateup
- Donald C Rio
- Dirk Hockemeyer
- Frank Soldner
Albert Einstein College of Medicine, Yeshiva University (Internal research support from the Department of Neuroscience)
- Frank Soldner
Siebel Stem Cell Institute (Fellow)
- Helen S Bateup
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Li et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Highly efficient generation of isogenic pluripotent stem cell models using prime editing
eLife 11:e79208.
https://doi.org/10.7554/eLife.79208
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