Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.

CRISPR/Cas9 系统中引导 RNA 的表达通常需要使用 RNA 聚合酶 III 启动子，该启动子不具有细胞类型特异性。在 gRNA 侧翼添加自切割核酶基序以创建自切割 gRNA 克服了这一限制。在这里，我们使用自切割 gRNA 在特定肝细胞位点创建药物可选择的基因编辑事件。重组腺相关病毒载体靶向白蛋白基因座，具有无启动子自切割 gRNA 以产生耐药性，与治疗性转基因顺式连接。两者的基因表达都依赖于靶基因座的同源重组。对精确编辑的肝细胞进行体内药物选择可以使基因编辑的细胞扩增 30 倍以上，并达到人类因子 9转基因的治疗水平。重要的是，在靶向较弱的肝细胞基因后，也可以实现自切割 gRNA 表达。我们得出的结论是，自切割 gRNA 是一个强大的系统，可以在治疗性基因编辑应用中实现细胞类型特异性的体内耐药性。