Advanced In vivo Use of CRISPR/Cas9 and Anti-sense DNA Inhibition for Gene Manipulation in the Brain

Abstract

Gene editing tools are essential for uncovering how genes mediate normal brain–behavior relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene editing technology now allows neuroscientists unprecedented access to edit the genome efficiently. Although many important tools have been developed, here we focus on approaches that allow for rapid gene editing in the adult nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques. CRISPR/Cas9 is a flexible gene editing tool, allowing the genome to be manipulated in diverse ways. For instance, CRISPR/Cas9 has been successfully used to knockout genes, knock-in mutations, overexpress or inhibit gene activity, and provide scaffolding for recruiting specific epigenetic regulators to individual genes and gene regions. Moreover, the CRISPR/Cas9 system may be modified to target multiple genes at one time, affording simultaneous inhibition and overexpression of distinct genetic targets. Although many of the more advanced applications of CRISPR/Cas9 have not been applied to the nervous system, the toolbox is widely accessible, such that it is poised to help advance neuroscience. Anti-sense nucleotide-based technologies can be used to rapidly knockdown genes in the brain. The main advantage of anti-sense based tools is their simplicity, allowing for rapid gene delivery with minimal technical expertise. Here, we describe the main applications and functions of each of these systems with an emphasis on their many potential applications in neuroscience laboratories.

Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

“…Given such advances in gene targeting, the method originally developed by the three pioneering Nobel Laureates, Capecchi, Smithies and Evans, will likely withstand the test of times. Nevertheless, it is also likely that in the future new recombinant DNA methods, including the TALEN (transcription activator-like effector nuclease) and the Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (clustered regularly interspaced short palindromic repeats) gene targeting systems ( Boettcher and McManus, 2015 ; Pu et al, 2015 ; also see current special topic paper by Walters et al, 2016 ), will gain increasingly strong roles in the genetic analysis of brain function and behavior. One reason why these newly developed techniques may become more frequently used at the expense of classical gene targeting is that even though true-and-tried, the ES cell-based gene targeting method is more time consuming, labor intensive and expensive than many of the recently developed genome editing tools.…”

Common therapeutic strategies for prion and Alzheimer’s diseases

“…Thanks to recent improvements, the CRISPR/Cas9 system has become highly efficient and specific (Zhang et al, 2014), and it has recently been employed to produce PRNP knockout mouse epithelial NMuMG cells (Mehrabian et al, 2014). Important challenges remain in the clinical application of CRISPR/Cas9 and other gene-editing technologies to target neuronal proteins, related to their delivery (Walters et al, 2015). The use of viral carriers, such as lentiviral, adenoviral or adeno-associated vectors, presents technical and theoretical challenges, mainly due to the difficulty of precisely controlling expression, and avoiding unwanted insertional mutagenesis effects.…”

Progress With Nonhuman Animal Models of Addiction

“…The new gene editing methods (e.g., CRISPR/Cas9) have already been used to manipulate multiple genes at once (Shalem et al, 2015;Walters et al, 2015), and such use seems likely to spread. Propositions about the role of specific genetically modulated pathways could then be fairly powerfully tested.…”