Xinhe Zheng

Abstract Systematic analysis of gene function across diverse cell types in vivo is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell’s perturbation in high-throughput single-cell assays. Leveraging AAV’s versatile cell type tropism and high labeling capacity, we expanded the resolution and scale of in vivo CRISPR screens: allowing phenotypic analysis at single-cell resolution across a multitude of cell types in the embryonic brain, adult brain, and peripheral nervous system. We undertook extensive tests of 86 AAV serotypes, combined with a transposon system, to substantially amplify labeling and accelerate in vivo gene delivery from weeks to days. Using this platform, we performed an in utero genetic screen as proof-of-principle and identified pleiotropic regulatory networks of Foxg1 in cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct networks essential for cell fate specification. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% (mediated by lentivirus), and achieve analysis of over 30,000 cells in one experiment, thus enabling massively parallel in vivo Perturb-seq. Compatible with various perturbation techniques (CRISPRa/i) and phenotypic measurements (single-cell or spatial multi-omics), our platform presents a flexible, modular approach to interrogate gene function across diverse cell types in vivo , connecting gene variants to their causal functions.

Tremendous progress has been made in identifying genetic variants associated with neurodevelopmental disorders (NDDs), particularly autism spectrum disorder (ASD). However, the extensive (and growing) lists of associated genetic variants have led to a bottleneck in understanding the function of these genetic changes. To overcome this, functional genomics approaches-including high-throughput and high-content screens, in vivo Perturb-seq, and multiomics profiling-are being deployed across cellular and animal models at scale. Here, we first discuss recent findings on NDDs gleaned from human genetics studies. We then review recent technological advances and findings from functional neurogenomics in the context of ASD and other NDDs. Finally, we discuss how these methods might be applied in the future to refine efforts to identify convergent mechanisms impacted by multiple disease-associated genetic variants, as well as how they can advance the development of new therapeutic strategies.

Abstract DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding homology search in cells is the lack of tools to monitor this process. We developed RA D51 p roximity id entification seq uencing (RaPID-seq), a sensitive method that marks all candidate templates searched by RAD51. We find that HDR is hierarchical, such that DSB proximity determines template candidacy and subsequent recombination is unlocked by DSB/template homology. Sequences that lie outside the proximal window are not efficiently searched, even if identical in sequence. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.

TNBC is an aggressive subtype of breast case which is hard to treat due to the high degree of heterogeneity and higher frequency of metastasis. Luminal progenitor cells (LPs) were previously suggested to play a role in the metastasis in TNBC. In this study, we use a publicly available scRNA-Seq dataset (GSE118389) to address the differences in transcriptional signatures of LPs and luminal epithelial cells in TNBC patients with and without lymph node involvement. When comparing the expression level of genes, we found there is a obvious difference in luminal epithelial cells between TNBC patients with and without lymph node involvement.