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Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

Hanqin Li1,2,3, Oriol Busquets3,4, Yogendra Verma1,3

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

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|September 7, 2022
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Summary

Prime editing (PE) offers a simpler way to create human pluripotent stem cell (hPSC) disease models. Optimized mRNA delivery significantly boosts PE efficiency in hPSCs for precise gene editing.

Keywords:
disease modelsgeneticsgenome engineeringgenomicshPSCshumanparkinson's diseaseprime editingregenerative medicinestem cells

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Area of Science:

  • Biotechnology
  • Genetics
  • Stem Cell Biology

Background:

  • Prime editing (PE) is a novel genome engineering technology.
  • PE utilizes a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and a PE guide RNA (pegRNA).
  • PE enables precise introduction of designer mutations via reverse transcription from the pegRNA template.

Purpose of the Study:

  • To systematically compare the editing efficiencies of PE with conventional gene editing methods in human pluripotent stem cells (hPSCs).
  • To investigate factors limiting PE efficiency in hPSCs and optimize delivery methods.
  • To demonstrate the application of optimized PE for creating disease models, including Parkinson's disease.

Main Methods:

  • Systematic comparison of PE editing efficiencies against homology-directed repair in hPSCs.
  • Stable integration of nCas9-RT into hPSCs to assess editing capabilities.
  • Optimization of PE component delivery modalities, focusing on mRNA delivery with chemically modified RNAs.
  • Repeated application of optimized PE for gene correction and introduction of familial mutations.

Main Results:

  • PE demonstrated higher efficiency and precision than conventional methods in hPSCs, particularly for heterozygous editing.
  • Stable nCas9-RT expression in hPSCs achieved efficiencies comparable to cancer cells.
  • Optimized mRNA delivery of PE components, including modified RNAs, enhanced editing efficiencies up to 13-fold.
  • Repeated mRNA-based delivery achieved over 60% editing efficiency, successfully correcting/introducing Parkinson's disease mutations.

Conclusions:

  • Prime editing is a highly efficient and precise tool for generating hPSC-based disease models.
  • Optimizing delivery methods, particularly using mRNA and modified RNAs, is crucial for maximizing PE efficiency in hPSCs.
  • This optimized PE system provides a powerful platform for studying genetic diseases like Parkinson's and developing therapeutic strategies.