Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
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DOI：
10.1038/s41467-022-35097-5
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发表时间：
2022-11-30
影响因子：
16.6
通讯作者：
Grompe, Markus
中科院分区：
文献类型：
--
作者：
Tiyaboonchai, Amita;Vonada, Anne;Posey, Jeffrey;Pelz, Carl;Wakefield, Leslie;Grompe, Markus
在CRISPR/Cas9系统中表达指导RNA通常需要使用RNA聚合酶III启动子，其不是细胞类型特异性的。用自切割核酶基序侧接gRNA以产生自切割gRNA克服了这一限制。在这里，我们使用自切割gRNA在特定的肝细胞基因座中创建药物选择性基因编辑事件。靶向白蛋白基因座的具有无启动子自切割gRNA以产生耐药性的重组腺相关病毒载体与治疗性转基因顺式连接。两者的基因表达都依赖于同源重组进入靶基因座。用于精确编辑的肝细胞的体内药物选择允许基因编辑的细胞扩增>30倍，并导致人因子9转基因的治疗水平。重要的是，在靶向弱肝细胞基因后也实现了自切割gRNA表达。我们得出结论，自切割gRNA是一种强大的系统，能够实现细胞类型特异性体内耐药性，用于治疗性基因编辑应用。在这里，作者表明，rAAV载体精确整合到染色体靶基因座中导致自切割指导RNA的表达。这种重组事件产生了对肝毒性化合物的遗传抗性，并能够使用选择药物在体内扩增基因编辑的肝细胞。
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. Here, authors show that precise integration of an rAAV vector into a chromosomal target locus results in expression of a self-cleaving guide RNA. This recombination event creates genetic resistance to a hepatotoxic compound and enables in vivo expansion of gene-edited hepatocytes using a selection drug.
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影响因子：
64.8
作者：
Barzel, A.;Paulk, N. K.;Shi, Y.;Huang, Y.;Chu, K.;Zhang, F.;Valdmanis, P. N.;Spector, L. P.;Porteus, M. H.;Gaensler, K. M.;Kay, M. A.
通讯作者：
Kay, M. A.
影响因子：
3.9
作者：
Iyanagi, Takashi;Xia, Chuanwu;Kim, Jung-Ja P.
通讯作者：
Kim, Jung-Ja P.
影响因子：
30.8
作者：
GROMPE, M;LINDSTEDT, S;FINEGOLD, M
通讯作者：
FINEGOLD, M
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
影响因子：
16
作者：
Clarke, Ryan;Terry, Alexander R.;Pennington, Hannah;Hasty, Cody;MacDougall, Matthew S.;Regan, Maureen;Merrill, Bradley J.
通讯作者：
Merrill, Bradley J.