Abstract
CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system. Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene Brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.
Significance We report a neuron-optimized CRISPR/dCas9 activation (CRISPRa) system that produces robust and specific upregulation of targeted genes in neurons both in vitro and in vivo. This system effectively drives expression at many gene targets, provides titratable gene expression, is capable of simultaneously targeting multiple genes at once, and successfully targets individual transcript variants arising from a complex, multi-promoter gene. This molecular tool enables advances in our ability to control gene expression profiles in the brain and will enable expansion of gene regulatory investigations to model systems that have not typically been used to explore genetic control of neuronal function.
Footnotes
The authors report no conflict of interest.
NIH grants DA039650, DA034681, and MH114990 (J.J.D.), DA042514 (K.E.S.), MH112304 (S.V.B), DA041778 (F.A.S.). L.I. is supported by the Civitan International Research Center at UAB. Additional assistance to J.J.D. was provided by the UAB Pittman Scholars Program.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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