Nature Neuroscience recently published a study demonstrating that the transcription of multiple genes in the brain can be simultaneously activated using CRISPR–dCas9-activator transgenic mice. This work was performed by researchers in Dr. YANG Hui’s lab at the Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, and Dr. HUANG Pengyu's lab at ShanghaiTech University. This work generated a transgenic mouse expressing an improved dCas9 activation system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. This system provides a flexible platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.
The ability to control the expression of any genes at will has been a dream of many biologists. CRISPR/Cas9, known as a "molecular scissor", cleaves the DNA double-strand with high specificity. Recently, acatalytically dead Cas9 (dCas9) was developed as a powerful alternative. Targeted activation of endogenous genes can be achieved by fusing dCas9 with transcriptional activators. dCas9-based transcriptional activation has many advantages over traditional overexpression methods. It is not limited by the size of an inserted open reading frame (ORF) sequence in the viral expression vectors, and therefore multiplex activation can be easily achieved. Moreover, it can accurately model the expressions of the complex transcript isoform variances. dCas9-mediated activation has been verified and widely used in different cell lines in vitro. However, in vivo applications of dCas9-activator remain unverified.
In this study, researchers designed an improved activator SPH which showed more potent activation efficiency in both human and mouse cell lines, compared with different representative second-generation activators. Then, the researchers generated a Cre-dependent SPH transgenic mouse, and showed that genes and IncRNAs could be activated in primary cells, after being infected by a lentivirus, expressing sgRNAs and Cre recombinase. To determine the feasibility of using SPH mice for inducing transcriptional activation in vivo, sgRNAs plasmids were delivered to the liver by a hydrodynamic injection, demonstrating that genes could be efficiently upregulated in vivo. The metabolic zonation of the liver was remodeled via a targeted activation of the Dkk1 gene, which encoded a Wnt antagonist. The researchers further showed that mature astrocytes in the midbrain can be converted into functional neurons by injecting AAV targeting three previously described neurogenic transcription factors: Ascl1, Neurog2 and Neurod1. This confirmed that SPH transgenic mice can be used to modulate neuronal functions in vivo. Finally, the researchers injected sgRNA arrays targeting ten genes or ten genetic elements, including eight genes and two lncRNAs into the brain. As expected, most of the targets were potently upregulated. Collectively, these results highlight the advantages and potential of using SPH mice to modulate complex genetic networks in the intact brain.
Their work entitled “In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice” was published online in Nature Neuroscience on January 15, 2017. Haibo Zhou, Junlai Liu, Changyang Zhou, Ni Gao, Zhiping Rao, He Li are the primary authors with equal contribution. This work was supported by ShanghaiTech University, the CAS Strategic Priority Research Program, the MoST863 Program, NSFC grants, China Youth Thousand Talents Program, the Breakthrough Project of the Chinese Academy of Sciences, and the Ministry of Science and Technology of China.
Graphic Abstract: This study developed an improved activator SPH and generated a Cre-dependent SPH transgenic mouse. The researchers demonstrated that SPH transgenic mice could be used to remodel the metabolic zonation of the liver and directly convert astrocytes into functional neurons, while simultaneously activating multiple genetic elements in the brain.
Nature Neuroscience advance online publication 15 January 2018: 10.1038/s41593-017-0060-6