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

Reviewer #1 (Public review):

Summary:

This is an interesting manuscript by Kirk and colleagues describing a highly valuable knock-down system that leverages CRISPRi in order to further elucidate the role of the Kruppel-Like Factor (KLF) transcription factor family in regulating the maturation of postnatal cortical projection neurons. The authors firstly use RNA-Seq and ATAC-Seq data in order to identify the KLF TF family as a potential regulator of cortical neuron maturation in the postnatal brain and subsequently knock down four KLF family members; KLF9, KL13, KLF6 and KLF7, in order to ascertain the functions of specific KLF genes in the developing cortex. The described CRISPRi knock down strategy is highly robust and penetrant as evidenced by a KD efficiency > 95% (assessed by both qPCR and single molecule FISH) and demonstrates that KLF6 and KLF7 play an activating role in driving the expression of target genes relating to axonal growth whereas KLF9 and 13 play a repressive role that inhibits the expression of overlapping gene targets. Together, the authors propose a model where the KLF TF family acts as a regulatory "switch" from activation to repression in the postnatal cortex as a mechanism to control a shift in projection neuron function from axonal growth to circuit refinement. The findings and conclusions of the manuscript offer a valuable contribution to the field of postnatal cortical development and further our understanding of the regulatory mechanisms that govern neuron maturation.

The conclusions of this manuscript are generally supported by the data, but some aspects of the data collection and analysis require some further clarification. Specifically:

(1) The authors comprehensively assess the molecular effects of KLF TF knock-down, however, the authors do not deeply address the cellular effects of these knock-downs. The authors conclude that knockdown of KLF6/7 and KLF9/13 cause downregulation and upregulation, respectively, of a common set of genes involved in cytoskeletal or axon regulation such as Tubb2 and Dpysl3. How is the morphology of the cells affected by these knockdowns? For example, does KLF9/13 knockdown cause neurite/axonal outgrowth? The authors should perform some basic experiments to assess changes in cell morphology following KLF TF KD. This is the one key point that needs addressing, in my opinion.

(2) The authors identify 374 DEGs in P10 Klf6/7 KD neurons and 115 DEGs at P20 (figure 6B). Have the authors looked to see what proportion of these DEGs are upregulated in the KLF9/13 KDs in order to get a more global understanding of the degree of overlap in the genes regulated by the KLF family members? Along similar lines, the authors later indicate that there are 144 shared targets between the KLF activator and repressor pairs (Figure 7C). What percentage does this represent of the total number of DEGs between the KLF pairs. This could further illustrate the degree to which the KLF pairs regulate the same set of genes. If it is already indicated in the manuscript, it should be made a bit more clear to the reader.

(3) Figures 5B and 6D2 are very interesting as they relate the changes in gene expression over time in neurons from P2 to P30 to the functions of KLF9/13 and KLF6/7, respectively. I would be curious to see how these two forms of analyses overlap with one another. For example, in Figure 6D2, where would the KLF9/13 upregulated genes fall on the plot shown in Figure 6D2? And would those overlapping genes fit a similar correlation?

(4) Figure 7E shows expression levels of shared KLF TF targets in control or KD conditions. Interestingly, the expression of Tubb2b, shows higher expression in ScrGFP P10 when compared to KLF9/13 P20, suggesting that derepression of KLF9/13 does not fully restore the expression level of Tubb2b seen at P10. This may suggest that other repressive regulators may be involved in the downregulation of Tubb2b from P10 to P20. Can the authors further comment on this, perhaps in the discussion, and speculate if there are other regulatory factors at play that may be controlling some of the shared targets by KLF6/7 and KLF9/13?

Reviewer #2 (Public review):

Summary:

Kirk et al. use RNA-Seq and CRISPRi to provide evidence that KLF family transcription factors regulate postnatal neuronal maturation of pyramidal neurons. The genetic programs regulating postnatal neuronal maturation are not well understood. The authors first analyzed chromatin accessibility and gene expression data from layer 4 and 6 pyramidal neurons and found that KLF TFs are predicted regulators of postnatal neuronal maturation. They then use CRISPRi knockdown and find that KLF activators first activate genes and then this is followed by KLF repressors repressing genes. Interestingly, some genes, such as those with cytoskeletal functions, are shared targets of KLF activators and repressors.

Strengths:

The study is well-executed and the paper is well-written. A major strength of this study is the application of state-of-the-art transgenic approaches. The CRISPRi approach used to knock down multiple KLFs is compelling. The genomic data generated appears to be high quality and is carefully analyzed. The presented findings provide important insights into the genetic programs that regulate postnatal maturation in cortical pyramidal neurons. The discovery that KLF family activators/repressors regulate gene expression changes during this critical step of neuronal development fills an important gap in the field.

Weaknesses:

A limitation of the current study is that the functional importance of KLF for postnatal neuronal maturation is unclear. Although the authors find that KLFs regulate some of the gene expression changes during postnatal neuronal maturation, it is still unclear whether such gene expression changes mediate the postnatal changes in morphology and physiology. While beyond the scope of the current study, future studies should investigate the contributions of KLFs on postnatal morphological and physiological changes.

Reviewer #3 (Public review):

Summary:

In their manuscript "Multiplexed CRISPRi Reveals a Transcriptional Switch Between KLF Activators and Repressors in the Maturing Neocortex", Kirk and colleagues seek to dissect the developmentally regulated pan-neuronal gene programs that control the postnatal maturation of cortical neurons. For this, the authors analyzed newly generated and existing RNA-seq and ATAC-seq of Layer 4 and Layer 6 cortical pyramidal neurons at postnatal day 2 (P2) and day 30 (P30), and identified thousands of shared developmentally regulated genes and genomic (promoter) regions, including genes involved in axon growth (tend to be downregulated) and synaptic function (tend to be upregulated). Motif enrichment analysis of promoters of differentially regulated genes revealed a strong presence of KLF/Sp family binding motifs, pointing to Krüppel-Like Factors (KLFs) as key transcriptional regulators of cortical maturation. Expression profiling showed a developmental switch from activating KLFs (Klf6, Klf7) expressed neonatally to repressive KLFs (Klf9, Klf13) upregulated during maturation. Using an elegant in vivo multiplexed CRISPR interference (CRISPRi) system, the authors achieved efficient, cell-type-specific knockdown of these TFs and showed that Klf9 and Klf13 repress a set of genes that includes cytoskeletal regulators such as Tubb2b, Dpysl3, and Rac3. Conversely, Klf6 and Klf7 promoted the expression of these same genes in the early postnatal period, and their knockdown led to reduced expression of these genes, particularly at P10 when their activating influence is strongest. Since promoters of shared KLF targets were enriched for KLF/Sp motifs but showed little change in chromatin accessibility, the authors propose a model in which distinct KLF family members function either as transcriptional repressors and activators that compete at constitutively accessible promoters and thereby act as a developmental transcriptional switch that coordinates the downregulation of axon growth programs and upregulation of synaptic maturation genes during cortical development.

Strengths:

The study addresses an interesting question and advances our understanding of the transcriptional regulation underlying postnatal cortical development. A major strength of the study lies in the innovative use of in vivo multiplexed CRISPR interference (CRISPRi), which allows for cell-type-specific, combinatorial knockdown of redundant TFs - this an elegant solution to a long-standing challenge in transcription factor research, and should be useful also for other neuroscience studies that require local and cell-type-specific gene loss-of-function. Also, the integration of RNA-seq and ATAC-seq across developmental time points provides a robust foundation for identifying direct targets of the KLF family, and the findings are reinforced by cross-species conservation and the identification of targets with clear neurodevelopmental relevance.

Weaknesses:

The major weakness of the study lies in its relatively narrow scope: the study focuses primarily on transcriptional mechanisms and largely lacks functional validation of the neuronal phenotypes that are predicted by the gene expression data (e.g. axonal morphology). For example, the authors analyzed the effects of KLF9/13 KD on the neurons' excitability and excitatory inputs, but did not assess the effects on inhibitory inputs and E/I-ratio or morphological parameters such as axonal length and axonal target fields - the manuscript would be strengthened considerably by such analyses (axonal projections could be analyzed e.g. via local injections of the gRNA AAVs and subsequent immunolabeling of brain sections). Similarly, the chromatin-based mechanisms underlying KLF activity remain relatively speculative, and the transcriptional mechanisms upstream of the KLFs remain unexplored (this could be addressed by analyzing existing datasets; see "Additional Point 1" below). Finally, the manuscript is too long (e.g., nearly five pages in the Discussion section are devoted to discussing various misregulated genes) and would benefit from presenting the Results and Discussion sections more concisely. However, despite these limitations, the paper offers an interesting model for a transcriptional switch during neuronal maturation in the cortex and establishes a powerful methodological framework for dissecting redundant gene networks in vivo.