Massively parallel characterization of regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Improving cellular diversity in human cerebellar organoids.

The developing human cerebellum has a greater diversity of progenitor types than that of the mouse, necessitating a human-based model for studying cerebellar development and disease. Atamian et al.1 developed a 3D organoid model of cerebellar development, which recapitulates many cell types found in the developing human cerebellum, including Purkinje-neuron-like cells.

Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing.

Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient's clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient's clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.

Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins.

Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.

Three-dimensional genome rewiring in loci with human accelerated regions.

Human accelerated regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with an automated pipeline and an alignment of 241 mammalian genomes. Combining deep learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs.

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Contribution of dorsal horn CGRP-expressing interneurons to mechanical sensitivity.

Primary sensory neurons are generally considered the only source of dorsal horn calcitonin gene-related peptide (CGRP), a neuropeptide critical to the transmission of pain messages. Using a tamoxifen-inducible CalcaCreER transgenic mouse, here we identified a distinct population of CGRP-expressing excitatory interneurons in lamina III of the spinal cord dorsal horn and trigeminal nucleus caudalis. These interneurons have spine-laden, dorsally directed, dendrites, and ventrally directed axons. As under resting conditions, CGRP interneurons are under tonic inhibitory control, neither innocuous nor noxious stimulation provoked significant Fos expression in these neurons. However, synchronous, electrical non-nociceptive Aß primary afferent stimulation of dorsal roots depolarized the CGRP interneurons, consistent with their receipt of a VGLUT1 innervation. On the other hand, chemogenetic activation of the neurons produced a mechanical hypersensitivity in response to von Frey stimulation, whereas their caspase-mediated ablation led to mechanical hyposensitivity. Finally, after partial peripheral nerve injury, innocuous stimulation (brush) induced significant Fos expression in the CGRP interneurons. These findings suggest that CGRP interneurons become hyperexcitable and contribute either to ascending circuits originating in deep dorsal horn or to the reflex circuits in baseline conditions, but not in the setting of nerve injury.

The ability to sense pain is critical to our survival. Normally, pain is provoked by intense heat or cold temperatures, strong force or a chemical stimulus, for example, capsaicin, the pain-provoking substance in chili peppers. However, if nerve fibers in the arms or legs are damaged, pain can occur in response to touch or pressure stimuli that are normally painless. This hypersensitivity is called mechanical allodynia. A protein called calcitonin gene-related peptide, or CGRP, has been implicated in mechanical allodynia and other chronic pain conditions, such as migraine. CGRP is found in, and released from, the neurons that receive and transmit pain messages from tissues, such as skin and muscles, to the spinal cord. However, only a few distinct groups of CGRP-expressing neurons have been identified and it is unclear if these nerve cells also contribute to mechanical allodynia. To investigate this, Löken et al. genetically engineered mice so that all nerve cells containing CGRP produced red fluorescent light when illuminated with a laser. This included a previously unexplored group of CGRP-expressing neurons found in a part of the spinal cord that is known to receive information about non-painful stimuli. Using neuroanatomical methods, Löken et al. monitored the activity of these neurons in response to various stimuli, before and after a partial nerve injury. This partial injury was induced via a surgery that cut off a few, but not all, branches of a key leg nerve. The experiments showed that in their normal state, the CGRP-expressing neurons hardly responded to mechanical stimulation. In fact, it was difficult to establish what they normally respond to. However, after a nerve injury, brushing the mice's skin evoked significant activity in these cells. Moreover, when these CGRP cells were artificially stimulated, the stimulation induced hypersensitivity to mechanical stimuli, even when the mice had no nerve damage. These results suggest that this group of neurons, which are normally suppressed, can become hyperexcitable and contribute to the development of mechanical allodynia. In summary, Löken et al. have identified a group of nerve cells in the spinal cord that process mechanical information and contribute to touch-evoked pain. Future studies will identify the nerve circuits that are targeted by CGRP released from these nerve cells. These circuits represent a new therapeutic target for managing chronic pain conditions related to nerve damage, specifically mechanical allodynia, which is the most common complaint of patients with chronic pain.