CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Single-nucleus sequencing reveals enriched expression of genetic risk factors in extratelencephalic neurons sensitive to degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by a progressive loss of motor function linked to degenerating extratelencephalic neurons/Betz cells (ETNs). The reasons why these neurons are selectively affected remain unclear. Here, to understand the unique molecular properties that may sensitize ETNs to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In both patients and unaffected individuals, we found significantly higher expression of ALS risk genes in THY1+ ETNs, regardless of diagnosis. In patients, this was accompanied by the induction of genes involved in protein homeostasis and stress responses that were significantly induced in a wide collection of ETNs. Examination of oligodendroglial and microglial nuclei revealed patient-specific downregulation of myelinating genes in oligodendrocytes and upregulation of an endolysosomal reactive state in microglia. Our findings suggest that selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitizing them to genetics and mechanisms of degeneration.

Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues.

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.

Loss of mouse Stmn2 function causes motor neuropathy.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.

Lipid-modified aminoglycoside derivatives for in vivo siRNA delivery.

Rationally designed siRNA delivery materials that are enabled by lipid-modified aminoglycosides are demonstrated. Leading materials identified are able to self-assemble with siRNA into well-defined nanoparticles and induce efficient gene knockdown both in vitro and in vivo. Histology studies and liver function tests reveal that no apparent toxicity is caused by these nanoparticles at doses over two orders of magnitude.

Remotely activated protein-producing nanoparticles.

The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.