Cerna-Chavez Rodrigo, Ortega-Gasco Alba, Baig Hafiz Muhammad Azhar, Ehrenreich Nathan, Metais Thibaud, Scandura Michael J, Bujakowska Kinga, Pierce Eric A, Garita-Hernandez Marcela
Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Int J Mol Sci. 2024 Dec 26;26(1):114. doi: 10.3390/ijms26010114.
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 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 and the c.6926A>C (p.H2309P) mutation in . 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.
碱基编辑（PE）是一种基于CRISPR的基因组工程工具，可用于生成基于人类诱导多能干细胞（hiPSC）的疾病模型。PE技术能够安全地将点突变、小片段插入和缺失（indel）引入基因组。它使用与逆转录酶（RT）融合的Cas9切口酶（nCas9）作为编辑器以及PE引导RNA（pegRNA），可在不产生双链断裂（DSB）的情况下精确引入所需编辑。在DNA中引入单链断裂（SSB）时，PE导致的脱靶或indel极少。效率低下可能会阻碍其在hiPSC中的应用，特别是当遗传背景排除了对多个pegRNA进行筛选时，必须采用其他策略来实现所需编辑。我们开发了一个PE平台，以高效生成孟德尔疾病的同基因模型。通过优化PE工作流程，我们以超过25%的效率在该基因中引入了c.25G>A（p.V9M）突变。使用我们优化的系统，我们生成了其他遗传性视网膜疾病（IRD）的同基因模型，包括该基因中的c.1481C>T（p.T494M）突变和该基因中的c.6926A>C（p.H2309P）突变。我们修改了hiPSC PE程序的几个决定因素，如质粒浓度、PE组件比例和递送方法设置，表明我们改进的工作流程提高了hiPSC编辑效率。
