Prime editing for functional repair in patient-derived disease models

Abstract

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin ( CTNNB1 ) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease ( ATP7B ). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

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Cited by papers we hold 8

• Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice · 2023
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• Efficient prime editing in mouse brain, liver and heart with dual AAVs · 2023
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• Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing · 2023
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• TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor · 2022
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• piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition · 2021
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• Enhanced prime editing systems by manipulating cellular determinants of editing outcomes · 2021
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• Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids · 2021
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• Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice · 2021
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