Methodologies and Challenges for CRISPR/Cas9 Mediated Genome Editing of the Mammalian Brain

Abstract

Neurons and glia are highly polarized cells with extensive subcellular structures extending over large distances from their cell bodies. Previous research has revealed elaborate protein signaling complexes localized within intracellular compartments. Thus, exploring the function and the localization of endogenous proteins is vital to understanding the precise molecular mechanisms underlying the synapse, cellular, and circuit function. Recent advances in CRISPR/Cas9-based genome editing techniques have allowed researchers to rapidly develop transgenic animal models and perform single-cell level genome editing in the mammalian brain. Here, we introduce and comprehensively review the latest techniques for genome-editing in whole animals using fertilized eggs and methods for gene editing in specific neuronal populations in the adult or developing mammalian brain. Finally, we describe the advantages and disadvantages of each technique, as well as the challenges that lie ahead to advance the generation of methodologies for genome editing in the brain using the current CRISPR/Cas9 system.

CRISPR/Cas9 unveils the dynamics of the endogenous µ‐opioid receptors on neuronal cells under continuous opioid stimulation

“…It is also possible to establish mutant mice that enable us to monitor MOP by applying similar genetic manipulation to fertilized eggs and obtaining offspring with the desired gene in a few months. 26 A previous study reported on post‐translational modifications of MOP by mass spectrometry using whole‐brain tissue. 27 Such mutant mice might provide information on the physiological regulation of endogenous MOP.…”

“…Using the aforementioned methods to those opioid receptor families might reveal their unknown physiological significance. It is also possible to establish mutant mice that enable us to monitor MOP by applying similar genetic manipulation to fertilized eggs and obtaining offspring with the desired gene in a few months 26 . A previous study reported on post‐translational modifications of MOP by mass spectrometry using whole‐brain tissue 27 .…”

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

“…Concerns regarding potential off-target edits caused by the CRISPR/Cas9 system have been voiced by multiple groups (Zhang et al 2015 ; Modell et al 2022 ). Other concerns such as in vivo payload delivery, immunogenicity, tissue system targeting, and unwanted genetic mosaicism (due to concurrently occurring cell division and genome editing) also remain prominent matters of contention in the scientific community (Mehta and Merkel 2020 ; Nishizono et al 2020 ; Gohil et al 2021 ; Modell et al 2022 ).…”

Open Frontiers in Neural Cell Type Investigations; Lessons From Caenorhabditis elegans and Beyond, Toward a Multimodal Integration

“…Combined expression studies and electron microscopy reconstructions in new model organisms, map tissue morphological and molecular characteristics to identify neural cell types ( Vergara et al, 2021 ). Additionally, CRISPR/Cas9-mediated genome editing delineates neural gene function in high resolution ( Nishizono et al, 2020 ; Fang et al, 2021 ). Individual researcher groups and consortia deliver large-scale profiling data and cell biology experimentalists are key to functionally dissect them.…”