dCas13-mediated translational repression for accurate gene silencing in mammalian cells.

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

Complex activity and short-term plasticity of human cerebral organoids reciprocally connected with axons.

An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits, we investigated an in vitro neural tissue model for inter-regional connections, in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids, suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition, optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro.

ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.