Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.
Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.
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DOI：
10.1093/nar/gkx154
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发表时间：
2017-06-20
影响因子：
14.9
通讯作者：
Schaffer DV
中科院分区：
文献类型：
--
作者：
Gaj T;Staahl BT;Rodrigues GMC;Limsirichai P;Ekman FK;Doudna JA;Schaffer DV
实现基因组编辑的全部潜力需要开发用于递送可编程核酸酶和供体模板以用于同源定向修复（HDR）的有效且广泛适用的方法。RNA引导的Cas9内切核酸酶可以作为与单一引导RNA（sgRNA）复合的纯化蛋白质引入细胞中。这样的核糖核蛋白（RNP）可以促进在与单链DNA寡核苷酸共递送之后经由HDR高保真引入单碱基取代。然而，将RNP与含有转基因的供体模板结合用于靶向基因添加已被证明具有挑战性，这反过来限制了RNP介导的基因组编辑工具箱的能力。在这里，我们证明了将RNP递送与天然重组腺相关病毒（AAV）供体载体相结合能够通过同源定向基因组编辑实现位点特异性基因插入。与传统的基于质粒的表达载体和供体模板相比，我们表明RNP和AAV供体递送的组合将基因添加的效率提高了12倍，使得能够创建谱系报告基因，其可用于在真实的时间内跟踪来自人成纤维细胞的纹状体神经元的转化。因此，这些结果说明了将核酸酶蛋白递送与AAV供体载体统一用于同源定向基因组编辑的潜力。
Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.