An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.
An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.
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DOI：
10.3389/fgeed.2020.00009
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发表时间：
2020
影响因子：
--
通讯作者：
Day JJ
中科院分区：
文献类型：
--
作者：
Duke CG;Bach SV;Revanna JS;Sultan FA;Southern NT;Davis MN;Carullo NVN;Bauman AJ;Phillips RA 3rd;Day JJ
遗传物质的表达支配着大脑的发育、分化和功能，需要对基因表达进行有针对性的操作，以了解基因功能对健康和疾病状态的贡献。尽管最近CRISPR/dCas9干扰（CRISPRi）技术的改进已经在选定的基因组位点上实现了靶向转录抑制，但将这些技术整合到非分裂神经元系统中仍然具有挑战性。此前，我们优化了双慢病毒表达系统，在有丝分裂后神经元中表达基于crispr的激活机制。在这里，我们使用了类似的策略来适应改进的dCas9-KRAB-MeCP2抑制系统，以实现神经元中强大的转录抑制。我们发现，由神经元选择性人突触蛋白启动子驱动的慢病毒递送dCas9-KRAB-MeCP2构建体可在原代大鼠神经元中实现转基因表达。接下来，我们展示了利用CRISPR sgRNAs靶向多种基因启动子的转录抑制，并展示了与现有的RNA干扰方法相比，该系统在神经元中对复杂脑源性神经营养因子（Bdnf）基因进行稳健的转录特异性操作的优势。我们的发现首次将这种改良的CRISPRi技术用于神经系统，有可能提高操纵神经系统中基因表达状态的能力。
The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.
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影响因子：
29
作者：
Bettencourt, Conceicao;Ryten, Mina;Forabosco, Paola;Schorge, Stephanie;Hersheson, Joshua;Hardy, John;Houlden, Henry
通讯作者：
Houlden, Henry
影响因子：
7.6
作者：
Hallock, Henry L.;Quillian, Henry M.;Martinowich, Keri
通讯作者：
Martinowich, Keri
影响因子：
7.2
作者：
Alberini, Cristina M.;Kandel, Eric R.
通讯作者：
Kandel, Eric R.
影响因子：
2.7
作者：
Campbell, Malcolm G.;Kohane, Isaac S.;Kong, Sek Won
通讯作者：
Kong, Sek Won
影响因子：
15.1
作者：
Eisener-Dorman, Amy F.;Lawrence, David A.;Bolivar, Valerie J.
通讯作者：
Bolivar, Valerie J.