Department CIBIO, Laboratory of Molecular Virology, University of Trento, Via Sommarive 9, 38123 Trento, Italy.
Imagine Institute, Laboratory of Chromatin and Gene Regulation During Development, Université de Paris, INSERM UMR 1163, Paris, France.
Mol Ther. 2023 Jul 5;31(7):2257-2265. doi: 10.1016/j.ymthe.2023.03.007. Epub 2023 Mar 10.
Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.
电穿孔 Cas9 核糖核蛋白（RNP）复合物具有防止脱靶切割和由核酸酶长期表达产生的潜在免疫反应的优势。然而，大多数工程化的高保真链球菌 Cas9（SpCas9）变体的活性低于野生型酶，并且与 RNP 递送不兼容。基于我们之前对 evoCas9 的研究，我们开发了一种适合 RNP 递送的高保真 SpCas9 变体。通过 K526D 取代的重组高保真 Cas9（rCas9HF）的编辑效率和精度与目前唯一可用的可作为 RNP 使用的高保真 Cas9（HiFi Cas9）的 R691A 突变体进行了比较。比较分析扩展到基因替代实验，其中两种高保真度与 DNA 供体模板结合使用，针对精确编辑产生不同比例的非同源末端连接（NHEJ）与同源定向修复（HDR）。分析表明，两种变体在整个基因组中具有不同的靶向能力，表现出不均匀的效率和精度。rCas9HF 的开发具有与当前在 RNP 电穿孔中使用的 HiFi Cas9 不同的编辑特征，为最高精度和高效应用增加了基因组编辑解决方案。
