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PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G&gt;A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C&gt;T (p.T494M) mutation in PRPF3 and the c.6926A&gt;C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency.<\/jats:p>","DOI":"10.3390\/ijms26010114","type":"journal-article","created":{"date-parts":[[2024,12,26]],"date-time":"2024-12-26T19:33:07Z","timestamp":1735241587000},"page":"114","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5446-5450","authenticated-orcid":false,"given":"Rodrigo","family":"Cerna-Chavez","sequence":"first","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alba","family":"Ortega-Gasco","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9370-1716","authenticated-orcid":false,"given":"Hafiz Muhammad Azhar","family":"Baig","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Ehrenreich","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thibaud","family":"Metais","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Scandura","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6809-9818","authenticated-orcid":false,"given":"Kinga","family":"Bujakowska","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric A.","family":"Pierce","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcela","family":"Garita-Hernandez","sequence":"additional","affiliation":[{"name":"Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"798","DOI":"10.1038\/s41436-018-0408-7","article-title":"Insights into genetics, human biology and disease gleaned from family based genomic studies","volume":"21","author":"Posey","year":"2019","journal-title":"Genet. 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