Multiplexed CRISPRi Reveals a Transcriptional Switch Between KLF Activators and Repressors in the Maturing Neocortex

Area of Science:

• Neuroscience
• Developmental Biology
• Molecular Biology

Background:

• Postnatal mammalian brain development involves significant neuronal changes, including loss of axon outgrowth capacity.
• Transcriptional mechanisms governing postnatal neuronal maturation are less understood than prenatal development.

Purpose of the Study:

• To investigate the transcriptional mechanisms regulating postnatal neuronal maturation in the mouse neocortex.
• To identify transcription factors involved in the transition from immature to mature neuronal properties.

Main Methods:

• Integrated chromatin accessibility and gene expression data from neocortical pyramidal neurons.
• Employed multiplexed CRISPR Interference (CRISPRi) for KLF family knockdown.
• Analyzed gene targets including Tubb2b and Dpysl3.

Main Results:

• Predicted and confirmed a role for Krüppel-Like Factor (KLF) transcription factors in regulating neonatally expressed genes.
• Identified a transcriptional switch involving KLF activators (Klf6, Klf7) and repressors (Klf9, Klf13).
• Demonstrated KLF paralog redundancy and the effectiveness of CRISPRi in overcoming it.

Conclusions:

• KLF family competition between activators and repressors regulates a key postnatal maturation program.
• This regulatory switch may explain the loss of intrinsic axon growth in maturing neurons.
• Facilitates the transition from axon growth to synaptic refinement for mature circuit stabilization.