Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. 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>A (p.V9M) mutation in the <i>NMNAT1</i> 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>T (p.T494M) mutation in <i>PRPF3</i> and the c.6926A>C (p.H2309P) mutation in <i>PRPF8</i>. 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.