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.

Prime editing enables precise genome editing in mouse liver and retina

“…4c, d). This highly specific editing by prime editor 3 is in line with recent results showing that off-target effects were not detectable when PE3 was used in mouse embryos 5 or human organoids 3 and is also compatible with the low frequency of off-target effects of PE3 in cultured mammalian cells 1,18 . (pOT1, .., pOT4) and those for the used sgRNA (sOT1, …, sOT4) in PE3-treated liver tissues.…”

“…Prime editing has been used in cultured mammalian cells 1,2 and organoids 3 , plants 4 , and mouse embryos 5 to introduce genetic changes in a targeted manner. However, the efficiency and precision of prime editing in postnatal mammals in vivo have not been determined.…”

APOE: The New Frontier in the Development of a Therapeutic Target towards Precision Medicine in Late-Onset Alzheimer’s

“…A study that used APOE to compare the technologies demonstrated a higher percentage of correction with the base editing approach compared to the conventional gene editing approach with less non-specific events [ 106 ]. A recent development in the CRISPR-Cas field, prime-editing [ 107 ], may offer a more precise and safe approach with much less undesired effects to correct the APOE e4 coding SNP.…”

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

“…The ability to precisely install or correct pathogenic mutations regardless of their composition makes prime editing an intriguing approach to perform somatic genome editing in model organisms to study disease processes or to utilize for therapeutic applications. Previous studies have used prime editing to recode loci in cultured cells 6 , plants 7 , stem cells 8,9 , and mouse zygotes 10,11 . However, PE delivery in adult animals has not yet been described.…”