AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

TY - JOUR

T1 - AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

AU - Deutsch, Matthias

AU - Günther, Anne

AU - Lerchundi, Rodrigo

AU - Rose, Christine R

AU - Balfanz, Sabine

AU - Baumann, Arnd

PY - 2021/2/4

Y1 - 2021/2/4

N2 - Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

AB - Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

KW - Animals

KW - CRISPR-Cas Systems/genetics

KW - Cells, Cultured

KW - Dependovirus/metabolism

KW - Electrophysiological Phenomena

KW - Gene Expression Regulation

KW - Gene Knockdown Techniques

KW - HEK293 Cells

KW - Hippocampus/cytology

KW - Humans

KW - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics

KW - Mice, Inbred C57BL

KW - Neurons/metabolism

KW - Protein Subunits/metabolism

KW - RNA Interference

KW - RNA, Messenger/genetics

KW - RNA, Small Interfering/metabolism

U2 - 10.3390/cells10020324

DO - 10.3390/cells10020324

M3 - SCORING: Journal article

C2 - 33557342

VL - 10

JO - CELLS-BASEL

JF - CELLS-BASEL

SN - 2073-4409

IS - 2

M1 - 324

ER -