Abstract
Balanced expression of ion channels in neurons is critical for maintaining healthy brain activity. Mutations in ion channel genes leading to neuropsychiatric diseases have been extensively studied.
However, little is known on how the expression of ion channel genes is transcriptionally regulated.
Previous bioinformatic screen using the assay for transposase-accessible chromatin with
sequencing(ATAC-seq) identified enriched binding motifs of the Kruppel-like factors (KLF) transcription
factor family within the promoters of certain ion channel genes. Furthermore, in an RNA-seq screen for
genes affected by activity deprivation in neurons, 2 KLF family members (6 and 9) were identified as
being 2-fold down-regulated by activity deprivation. The expression of two ion channels, Scn3a and
Cacna1e, are both potently upregulated during the same time. Mutations in those two ion channels are
known to play an important role in human epilepsy. Based on those observations, we hypothesized that
KLF6 and KLF9, are transcriptional repressors of ion channel genes, including Scn3a and Cacna1e. To
test this, we utilized CRISPR interference to knock down KLF6 and 9 in mouse cortical excitatory
neurons. The RT-qPCR data from the knockdown samples confirmed high knockdown efficiency (over
90%) for both targets and RNA-seq revealed many differentially expressed genes due to the
knockdown(s). To further understand the potential role of KLFs regulating ion channel gene expression,
we knocked down KLF13 and a combination of KLF9 and 13. Notably, an off-target effect that was
dependent on the number of guide RNA delivered was also observed. Overall, our results confirmed the
effectiveness of the development of CRISPRi technology, especially its integration into transgenic mice
lines, and offers a novel way to investigate gene expression and regulation in a cell-type-specific manner.
We also report an off-target effect of the CRISPRi system that may require future investigation to better
understand.