RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.
RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.
复制标题
DOI：
10.1038/s41467-018-04048-4
复制
发表时间：
2018-04-26
影响因子：
16.6
通讯作者：
Gersbach CA
中科院分区：
文献类型：
--
作者：
Thakore PI;Kwon JB;Nelson CE;Rouse DC;Gemberling MP;Oliver ML;Gersbach CA
CRISPR-Cas9转录阻遏物已成为体外破坏基因调控的强大工具，但尚未适用于成年动物模型中的全身递送。在这里，我们描述了与腺相关病毒（AAV）递送相容的基于金黄色葡萄球菌Cas9的阻遏物（dSaCas 9 KRAB）。为了评估dSaCas 9 KRAB在体内基因沉默的功效，我们在成年小鼠的肝脏中沉默了胆固醇水平的调节剂Pcsk 9的转录。系统性施用表达dSaCas 9 KRAB和靶向Pcsk 9的指导RNA（gRNA）的双载体AAV 8系统导致血清Pcsk 9和胆固醇水平显著降低。尽管宿主对dSaCas 9 KRAB表达有中度应答，但Pcsk 9抑制在单次处理后维持24周，证明了dSaCas 9 KRAB在有丝分裂后组织中长期基因沉默的潜力。体内可编程基因沉默使研究能够将基因调控与复杂表型联系起来，并扩展了CRISPR-Cas9扰动工具箱，用于基础研究和基因治疗应用。使用CRISPR-Cas9抑制基因转录已经在体外实现，但不能用于递送到成年动物模型中。在这里，作者使用AAV 8将转录抑制因子dSaCas 9 KRAB传递给胆固醇调节因子Pcsk 9，并在小鼠中显示了长达24周的抑制和降低的胆固醇水平。
CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications. Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.
登录
查看更多内容
影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
64.5
作者：
Liao HK;Hatanaka F;Araoka T;Reddy P;Wu MZ;Sui Y;Yamauchi T;Sakurai M;O'Keefe DD;Núñez-Delicado E;Guillen P;Campistol JM;Wu CJ;Lu LF;Esteban CR;Izpisua Belmonte JC
通讯作者：
Izpisua Belmonte JC
影响因子：
158.5
作者：
Cohen, JC;Boerwinkle, E;Hobbs, HH
通讯作者：
Hobbs, HH