A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

eNeuro. 2019 Mar 7;6(1):ENEURO.0495-18.2019. doi: 10.1523/ENEURO.0495-18.2019. eCollection 2019 Jan-Feb.

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 (CNS). 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.

Keywords: Bdnf; CRISPR; epigenetics; gene expression; transcription.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / metabolism
  • Brain-Derived Neurotrophic Factor / genetics
  • Brain-Derived Neurotrophic Factor / metabolism
  • CRISPR-Cas Systems*
  • Cell Adhesion Molecules, Neuronal / metabolism
  • Cell Line, Tumor
  • Extracellular Matrix Proteins / metabolism
  • Gene Expression Regulation*
  • Genetic Techniques*
  • Male
  • Nerve Tissue Proteins / metabolism
  • Neurons / cytology
  • Neurons / metabolism*
  • Primary Cell Culture
  • Random Allocation
  • Rats, Sprague-Dawley
  • Reelin Protein
  • Serine Endopeptidases / metabolism
  • Transcription, Genetic
  • Transcriptome

Substances

  • Bdnf protein, rat
  • Brain-Derived Neurotrophic Factor
  • Cell Adhesion Molecules, Neuronal
  • Extracellular Matrix Proteins
  • Nerve Tissue Proteins
  • Reelin Protein
  • Serine Endopeptidases