期刊
ELIFE
卷 11, 期 -, 页码 -出版社
eLIFE SCIENCES PUBL LTD
DOI: 10.7554/eLife.79208
关键词
hPSCs; prime editing; disease models; parkinson's disease; genome engineering; Human
类别
资金
- Aligning Science Across Parkinson's [ASAP-000486]
- Albert Einstein College of Medicine, Yeshiva University
- National Cancer Institute [P30 CA013330]
- Siebel Stem Cell Institute
- Chan Zuckerberg Biohub
- National Institutes of Health, Office of Strategic Coordination [DP2 CA239597]
- National Human Genome Research Institute [R01 HG012227]
Recent development of prime editing technology has the potential to simplify the generation of human pluripotent stem cell-based disease models. It is more efficient and precise than conventional gene editing methods, particularly in generating disease-associated mutations. By optimizing the delivery modalities, editing efficiency can be further improved.
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 multicomponent 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 of site-specific nuclease-induced double-strand breaks. 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 improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. 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.
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