The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.

中文翻译：

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诱导肝再生增强 CRISPR/Cas9 介导的 1 型酪氨酸血症基因修复

突变附近的 CRISP/Cas9 切口增强了肝脏中同源重组的基因修复效率。在这项研究中，我们探索了旨在进一步增强遗传性酪氨酸血症模型中体内肝细胞基因修复的干预措施。采用双 AAV 系统：一种病毒携带化脓性葡萄球菌 Cas9 (SpCas9) 表达盒，另一种病毒携带 U6 启动子驱动的 sgRNA 和延胡索乙酰乙酸水解酶 ( Fah) 基因组 DNA 作为同源重组供体。在新生小鼠中，达到了 10.8% 的肝细胞的基因校正频率。成年小鼠的效率显着降低，约为 1.6%。为了确定肝细胞复制是否可以提高靶向频率，用甲状腺激素 T3 诱导细胞分裂。这将基因校正效率提高了一倍以上，达到 3.5% ( p  < 0.005)。为了确定 SpCas9 的传递是否存在速率限制，将基因修复 AAV 施用于 SpCas9 转基因小鼠。然而，这并没有显着增强基因修复。最后，我们测试了范可尼贫血 (FA) DNA 修复途径在肝细胞基因修复中是否重要。缺乏 FA 互补组 A 的新生小鼠的基因校正频率显着降低。Fanca ) 基因。总之，我们得出结论，肝细胞复制的药理学诱导以及DNA修复途径的操作可能是增强体内基因校正的有用策略。