CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

Abstract

The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo.

Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future

“…CRISPR/Cas9-mediated brain tumor modeling is likely to be more suitable for exploring the pathogenesis of brain tumors, as CRISPR/Cas9 platform is a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that are closely linked with brain tumors. Indeed, prior investigations supported the notion that the preparation of brain tumor modeling could be carried out via CRISPR knock out (CRISPR KO) technology [36, 44, 60-62], which were partly in line with our hypothesis that CRISPR technology was served to establish efficient knock-out brain tumor models. Recently, Zhang et al also emphasized that CRISPR/Cas9-mediated precise and efficient genome editing could help to deeply figure out the logic of neural circuits and disclose the mysteries of diverse neurological diseases including brain tumors [63].…”

Molecular Investigations of the Development and Diseases of Cerebral Cortex Folding using Gyrencephalic Mammal Ferrets

“…Ferrets have a venerable history in research because they have well-developed brain structures, such as cortical folds and the visual system, that mice do not have. [12][13][14][15] Because in utero electroporation has been widely used for expressing transgenes in the brain of rodents, [16][17][18] we tried to apply in utero electroporation to the ferret brain. Fortunately, we succeeded in establishing a rapid and efficient procedure of in utero electroporation for the ferret brain [7][8][9] (Fig.…”

“…[7][8][9] Furthermore, by combining in utero electroporation and the CRISPR/Cas9 system, we established gene knockout procedures for the cerebral cortex of mice and ferrets. 10,19,20) Our genetic manipulation techniques for ferrets enabled us to investigate the molecular mechanisms underlying the formation and malformation of cortical folds.…”

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

“…NHEJ functions in both dividing and nondividing cells, whereas HDR functions only in dividing cells910. CRISPR/Cas9-mediated gene knock-out in postmitotic neurons in vivo has been reported such as mouse cortical neurons, mouse hippocampal dentate gyrus granule neurons, and mouse cerebellar Purkinje cells13232425262728. CRSIPR/Cas9-mediated gene knock-in has been shown in dividing cells, but not in postmitotic neurons.…”