Applied & Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, Antwerp, Belgium.
Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
BMC Biol. 2023 Jul 13;21(1):156. doi: 10.1186/s12915-023-01646-7.
Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.
pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.
Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.
碱基编辑（Prime editing，PE）是最近出现的基因编辑技术，能够对基因组进行靶向修饰，包括单个碱基对的改变、小片段的插入和缺失。在过去几年中，PE 技术已经得到了多项改进，并在包括永生化细胞系、干细胞和动物模型在内的多种模型系统中进行了测试。尽管双切口 RNA（double nicking RNA，dncRNA）PE3 和 PE5 系统目前显示出最高的编辑效率，但由于在编辑位点产生了非预期的插入缺失或单核苷酸变异，其准确性降低。在这里，我们旨在通过生成由 EF-1α 启动子驱动的新型一体化（all-in-one，pAIO）质粒来改进单 RNA（single ncRNA，sncRNA）PE2 和 PE4max 系统，该启动子特别适合人类诱导多能干细胞（human-induced pluripotent stem cell，hiPSC）模型。
使用 pAIO-EF1α-PE2 和 pAIO-EF1α-PE4max 编辑电压门控钾通道基因 KCNQ2 和电压门控钠通道基因 SCN1A。使用 pAIO-EF1α-PE2 纠正了两种临床相关的突变，包括 HEK293T 细胞中的纯合截断 SCN1A R612*变体和患者来源的 hiPSC 中的杂合获得性功能 KCNQ2 R201C 变体。我们表明，sncRNA PE 在 hiPSC 中产生了可检测的编辑率，范围在 6.4%至 9.8%之间，在基于 GFP 的荧光激活细胞分选（fluorescence-activated cell sorting，FACS）细胞分选步骤后进一步增加到 41%。此外，我们表明，与低 GFP 表达群体相比，选择高 GFP 表达群体可将编辑效率提高多达 3.2 倍，表明不仅递送，而且每个细胞的 PE 酶和/或 pegRNA 的拷贝数对于有效编辑都很重要。在靶标基因座处引入靶基因侧翼沉默原间隔基序（protospacer-adjacent motif，PAM）缺失突变时，编辑效率没有提高。最后，使用 pAIO-EF1α-PE2 没有全基因组脱靶效应，在编辑位点附近也没有脱靶编辑活性，突出了 snc 碱基编辑器的准确性。
综上所述，我们的研究表明，EF-1α 驱动的 sncRNA pAIO-PE 质粒在 hiPSC 中的效率得到了提高，尤其是在 FACS 分选之后。优化这些 sncRNA PE 系统对于未来体内治疗的应用具有重要意义，因为准确性将是极其重要的。
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