A powerful new tool is available for neuroscience investigation into brain development, the mechanisms of memory and learning, and the role of brain dysregulation in neuropsychiatric diseases (lat. Morbo neuropsychiatric) like addiction, depression, schizophrenia and Alzheimer’s disease. This molecular biology tool - developed and validated by the University of Alabama at Birmingham researchers - is able to selectively and robustly turn on genes in neurons. It has been delivered to neurons growing in a dish, and more importantly, it has also been delivered to brain neurons in living adult rats. The tool can increase the expression of a single gene or the expression of multiple genes at the same time, and the amount of increased gene expression can be controlled.

This selective ability to increase gene expression - which means the gene makes more mRNA that codes for more protein - enables researchers to query the role of individual genes or groups of genes. Such queries are building blocks to learn how expression of different gene programs during development controls brain growth, wiring and the types of neurons created. For the adult brain, the tool can help explain how gene programs altered by neural activity and behavioral experience lead to adaptive behavior. The tool can also help investigate dysregulation of gene programs - many of which are implicated in neuropsychiatric diseases - that occur during brain development or in the fully developed adult brain.

The tool uses well-known CRISPR-based technology, which was packaged into lentiviruses for stable introduction into neurons. According to Jeremy Day, Ph.D., leader of the UAB research team, CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons have delayed incorporation of these tools in the central nervous system. Here, scientists demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation system capable of robust, modular and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems.

The CRISPR/Cas9 system is widely known as molecular scissors that can cut at a precise site in the genome to remove a gene or add a gene. An inactive form of CRISPR/Cas9 that is unable to cut DNA can be joined with transcriptional effectors to activate expression of a gene at a precise location. Jeremy Day and colleagues used this system, called CRISPRa. They packaged it into lentiviruses to allow stable expression of CRISPR machinery, allowing researchers to test its ability to regulate genes in neurons. The system includes a CRISPR-defective-Cas9 with activators and a single guide RNA that guides the CRISPR-dCas9 to a precise point on the genome.

One limitation of using CRISPRa in neurons is that promoters that are commonly used to drive molecular machinery in other types of cells were ineffective at driving CRISPR expression in neurons. To circumvent this, the UAB researchers selected a neuron-specific promoter, based on the promoter sequence for the human Synapsin, or SYN, gene, to express their CRISPRa tool in neurons. The UAB researchers then developed CRISPR single guide RNAs to target this system to a range of distinct genes, from small transcription factors to large extracellular proteins. They showed that this system significantly induced expression for each of these gene targets in primary rat neurons from the cortex, hippocampus and striatum of the brain.

Use of multiple single guide RNAs, called multiplexing, allowed tunable upregulation of single genes, or coordinated upregulation of multiple genes. The UAB teams also tested the system on a very complex brain gene, the brain-derived neurotrophic factor, or Bdnf. Bdnf is extremely difficult to study due to its complex transcriptional regulation, yet it is important for its central role in diverse processes such as neuronal differentiation and survival, dendritic growth, synaptic development, long-term potentiation, and memory formation. Bdnf has nine different promoter locations, and each leads to a variant mRNA transcript.

Due to this complexity, attempts to characterize distinct functional roles of individual Bdnf mRNAs in neurons have produced conflicting results, and currently available tools either lack the ability to selectively upregulate single Bdnf transcript variants or require cumbersome molecular cloning protocols to generate gene-specific targeting constructs. Jeremy Day and colleagues were able to use their CRISPR-dCas9 system to specifically target single promoters at two different sites on Bndf. Support came from National Institutes of Health grants; the Civitan International Research Center at UAB; and the UAB Pittman Scholars Program.