Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

Non-linear transcriptional responses to gradual modulation of transcription factor dosage.

Genomic loci associated with common traits and diseases are typically non-coding and likely impact gene expression, sometimes coinciding with rare loss-of-function variants in the target gene. However, our understanding of how gradual changes in gene dosage affect molecular, cellular, and organismal traits is currently limited. To address this gap, we induced gradual changes in gene expression of four genes using CRISPR activation and inactivation. Downstream transcriptional consequences of dosage modulation of three master trans-regulators associated with blood cell traits (GFI1B, NFE2, and MYB) were examined using targeted single-cell multimodal sequencing. We showed that guide tiling around the TSS is the most effective way to modulate cis gene expression across a wide range of fold-changes, with further effects from chromatin accessibility and histone marks that differ between the inhibition and activation systems. Our single-cell data allowed us to precisely detect subtle to large gene expression changes in dozens of trans genes, revealing that many responses to dosage changes of these three TFs are non-linear, including non-monotonic behaviours, even when constraining the fold-changes of the master regulators to a copy number gain or loss. We found that the dosage properties are linked to gene constraint and that some of these non-linear responses are enriched for disease and GWAS genes. Overall, our study provides a straightforward and scalable method to precisely modulate gene expression and gain insights into its downstream consequences at high resolution.

Enhancer-targeted CRISPR-Activation Rescues Haploinsufficient Autism Susceptibility Genes.

Autism Spectrum Disorder (ASD) is a highly heritable condition with diverse clinical presentations. Approximately 20% of ASD's genetic susceptibility is imparted by de novo mutations of major effect, most of which cause haploinsufficiency. We mapped enhancers of two high confidence autism genes - CHD8 and SCN2A and used CRISPR-based gene activation (CRISPR-A) in hPSC-derived excitatory neurons and cerebral forebrain organoids to correct the effects of haploinsufficiency, taking advantage of the presence of a wildtype allele of each gene and endogenous gene regulation. We found that CRISPR-A induced a sustained increase in CHD8 and SCN2A expression in treated neurons and organoids, with rescue of gene expression levels and mutation-associated phenotypes, including gene expression and physiology. These data support gene activation via targeting enhancers of haploinsufficient genes, as a therapeutic intervention in ASD and other neurodevelopmental disorders.

Low copy CRISPR-Cas13d mitigates collateral RNA cleavage.

While CRISPR-Cas13 systems excel in accurately targeting RNA, the potential for collateral RNA degradation poses a concern for therapeutic applications and limits broader adoption for transcriptome perturbations. We evaluate the extent to which collateral RNA cleavage occurs when Rfx Cas13d is delivered via plasmid transfection or lentiviral transduction and find that collateral activity only occurs with high levels of Rfx Cas13d expression. Using transcriptome-scale and combinatorial CRISPR pooled screens in cell lines with low-copy Rfx Cas13d, we find high on-target knockdown, without extensive collateral activity regardless of the expression level of the target gene. In contrast, transfection of Rfx Cas13d, which yields higher nuclease expression, results in collateral RNA degradation. Further, our analysis of a high-fidelity Cas13 variant uncovers a marked decrease in on-target efficiency, suggesting that its reduced collateral activity may be due to an overall diminished nuclease capability.

Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.

Essential transcription factors for induced neuron differentiation.

Neurogenins are proneural transcription factors required to specify neuronal identity. Their overexpression in human pluripotent stem cells rapidly produces cortical-like neurons with spiking activity and, because of this, they have been widely adopted for human neuron disease models. However, we do not fully understand the key downstream regulatory effectors responsible for driving neural differentiation. Here, using inducible expression of NEUROG1 and NEUROG2, we identify transcription factors (TFs) required for directed neuronal differentiation by combining expression and chromatin accessibility analyses with a pooled in vitro CRISPR-Cas9 screen targeting all ~1900 TFs in the human genome. The loss of one of these essential TFs (ZBTB18) yields few MAP2-positive neurons. Differentiated ZBTB18-null cells have radically altered gene expression, leading to cytoskeletal defects and stunted neurites and spines. In addition to identifying key downstream TFs for neuronal differentiation, our work develops an integrative multi-omics and TFome-wide perturbation platform to rapidly characterize essential TFs for the differentiation of any human cell type.