Julianne Jin

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.

Abstract Tau aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer’s disease and frontotemporal dementia. There are disease-causing variants of the tau-encoding gene, MAPT , and the presence of tau aggregates is highly correlated with disease progression. However, the molecular mechanisms linking pathological tau to neuronal dysfunction are not well understood. This is in part due to an incomplete understanding of the normal functions of tau in development and aging, and how the associated molecular and cellular processes change in the context of causal disease variants of tau. To address these questions in an unbiased manner, we conducted multi-omic characterization of iPSC-derived neurons harboring the MAPT V337M mutation or MAPT knockdown. RNA-seq, ATAC-seq, and phosphoproteomics revealed that both V337M mutation and tau knockdown perturbed levels of transcripts and phosphorylation of proteins related to axonogenesis or axon morphology. When we directly measured axonogenesis, we found that both MAPT V337M and MAPT knockdown caused decreased axon length. Surprisingly, we found that neurons with V337M tau had much lower tau phosphorylation than neurons with WT tau. CRISPR-based screens uncovered regulators of tau phosphorylation in neurons and found that factors involved in axonogenesis modified tau phosphorylation in both MAPT WT and MAPT V337M neurons. Intriguingly, the p38 MAPK pathway specifically modified tau phosphorylation in MAPT V337M neurons. We propose that V337M tau perturbs tau phosphorylation and axon morphology pathways that are relevant to the normal function of tau in development, which could contribute to previously reported cognitive changes in preclinical MAPT variant carriers.