Abstract：
The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.
摘要：
发现细菌防御机制，CRISPR-Cas9可以重新编程为基因编辑工具，彻底改变了基因编辑领域。CRISPR-Cas9可以在基因组内的特定靶向位点处引入双链断裂。随后的细胞内修复机制修复双链断裂,所述双链断裂可以导致基因敲除(通过非同源末端连接途径)或在DNA模板存在下通过同源定向修复导致的特定基因校正。对于后者，病理突变可以切除和修复。在患者中利用CRISPR-Cas9正在取得进展，方法是将其组分掺入非病毒递送载体中，这将保护它们免受过早降解并将它们递送到目标组织。在这里，CRISPR-Cas9可以以三种不同的货物的形式递送：质粒DNA，RNA或核糖核蛋白复合物(RNP)。我们和其他人最近表明，Cas9RNP可以有效地配制在脂质纳米颗粒（LNP）中，从而导致体外功能性递送。在这项研究中,我们比较了封装Cas9mRNA、sgRNA和HDR模板的LNP与含有Cas9-RNP和HDR模板的LNP。前者显示较小的粒径，在体外测定中，对报道HEK293T细胞和HEPA1-6细胞的降解酶有更好的保护作用和更高的基因编辑效率。在全身施用后,在雌性Ai9小鼠中另外测试两种制剂的生物分布和基因编辑效率。LNPmRNA传递Cas9主要保留在肝脏中，与LNP递送Cas9-RNP另外发现在脾和肺。最后,小鼠的基因编辑只能在LNP传递Cas9和sgRNA的过程中得出结论。这些LNP导致肝细胞中60%的基因敲除。由此得出结论,作为货物形式的mRNACas9的递送超过Cas9-RNP用于体外和体内基因编辑的CRISPR-Cas9的应用。
